Modified Human Growth Hormone

ABSTRACT

Modified growth hormone polypeptide and uses thereof are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional patent application60/638,616 filed Dec. 22, 2004 and U.S. provisional patent application60/727,996 filed Oct. 17, 2005, the specifications of which areincorporated herein in their entirety.

FIELD OF THE INVENTION

This invention relates to growth hormone polypeptides modified with atleast one non-naturally-encoded amino acid.

BACKGROUND OF THE INVENTION

The growth hormone (GH) supergene family (Bazan, F. Immunology Today 11:350-354 (1990); Mott, H. R. and Campbell, I. D. Current Opinion inStructural Biology 5: 114-121 (1995); Silvennoinen, O. and Ihle, J. N.(1996) S IGNALING BY THE HEMATOPOIETIC CYTOKINE RECEPTORS) represents aset of proteins with similar structural characteristics. Each member ofthis family of proteins comprises a four helical bundle, the generalstructure of which is shown in FIG. 1. While there are still moremembers of the family yet to be identified, some members of the familyinclude the following: growth hormone, prolactin, placental lactogen,erythropoietin (EPO), thrombopoietin (TPO), interleukin-2 (IL-2), IL-3,IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12 (p35 subunit), IL-13,IL-15, oncostatin M, ciliary neurotrophic factor, leukemia inhibitoryfactor, alpha interferon, beta interferon, gamma interferon, omegainterferon, tau interferon, epsilon interferon, granulocyte-colonystimulating factor (G-CSF), granulocyte-macrophage colony stimulatingfactor (GM-CSF), macrophage colony stimulating factor (M-CSF) andcardiotrophin-1 (CT-1) (“the GH supergene family”). Members of the GHsupergene family have similar secondary and tertiary structures, despitethe fact that they generally have limited amino acid or DNA sequenceidentity. The shared structural features allow new members of the genefamily to be readily identified. The general structures of familymembers hGH, EPO, IFNα-2, and G-CSF are shown in FIGS. 2, 3, 4, and 5,respectively.

One member of the GH supergene family is human growth hormone (hGH).Human growth hormone participates in much of the regulation of normalhuman growth and development. This naturally-occurring single-chainpituitary hormone consists of 191 amino acid residues and has amolecular weight of approximately 22 kDa. hGH exhibits a multitude ofbiological effects, including linear growth (somatogenesis), lactation,activation of macrophages, and insulin-like and diabetogenic effects,among others (Chawla, R., et al., Ann. Rev. Med. 34:519-547 (1983);Isaksson, O., et al., Ann. Rev. Physiol., 47:483-499 (1985); Hughes, J.and Friesen, H., Ann. Rev. Physiol., 47:469-482 (1985)).

The structure of hGH is well known (Goeddel, D., et al., Nature281:544-548 (1979)), and the three-dimensional structure of hGH has beensolved by X-ray crystallography (de Vos, A., et al., Science 255:306-312(1992)). The protein has a compact globular structure, comprising fouramphipathic alpha helical bundles, termed A-D beginning from theN-terminus, which are joined by loops. hGH also contains four cysteineresidues, which participate in two intramolecular disulfide bonds: C53is paired with C165 and C182 is paired with C189. The hormone is notglycosylated and has been expressed in a secreted form in E. coli(Chang, C., et al., Gene 55:189-196 (1987)).

A number of naturally occurring mutants of hGH have been identified.These include hGH-V (Seeburg, DNA 1: 239 (1982); U.S. Pat. Nos.4,446,235, 4,670,393, and 4,665,180, which are incorporated by referenceherein) and a 20-kDa hGH containing a deletion of residues 32-46 of hGH(Kostyo et al., Biochem. Biophys. Acta 925: 314 (1987); Lewis, U., etal., J. Biol. Chem., 253:2679-2687 (1978)). In addition, numerous hGHvariants, arising from post-transcriptional, post-translational,secretory, metabolic processing, and other physiological processes, havebeen reported (Baumann, G., Endocrine Reviews 12: 424 (1991)).

The biological effects of hGH derive from its interaction with specificcellular receptors. The hormone is a member of a family of homologousproteins that include placental lactogens and prolactins. hGH is unusualamong the family members, however, in that it exhibits broad speciesspecificity and binds to either the cloned somatogenic (Leung, D., etal., Nature 330:537-543 (1987)) or prolactin (Boutin, J., et al., Cell53:69-77 (1988)) receptor. Based on structural and biochemical studies,functional maps for the lactogenic and somatogenic binding domains havebeen proposed (Cunningham, B. and Wells, J., Proc. Natl. Acad. Sci. 88:3407 (1991)). The hGH receptor is a member of thehematopoietic/cytokine/growth factor receptor family, which includesseveral other growth factor receptors, such as the interleukin (IL)-3,-4 and -6 receptors, the granulocyte macrophage colony-stimulatingfactor (GM-CSF) receptor, the erythropoietin (EPO) receptor, as well asthe G-CSF receptor. See, Bazan, Proc. Natl. Acad. Sci. USA 87: 6934-6938(1990). Members of the cytokine receptor family contain four conservedcysteine residues and a tryptophan-serine-X-tryptophan-serine motifpositioned just outside the transmembrane region. The conservedsequences are thought to be involved in protein-protein interactions.See, e.g., Chiba et al., Biochim. Biophys. Res. Comm. 184: 485-490(1992). The interaction between hGH and extracellular domain of itsreceptor (hGHbp) is among the most well understood hormone-receptorinteractions. High-resolution X-ray crystallographic data (Cunningham,B., et al., Science, 254:821-825 (1991)) has shown that hGH has tworeceptor binding sites and binds two receptor molecules sequentiallyusing distinct sites on the molecule. The two receptor binding sites arereferred to as Site I and Site II. Site I includes the carboxy terminalend of helix D and parts of helix A and the A-B loop, whereas Site IIencompasses the amino terminal region of helix A and a portion of helixC. Binding of GH to its receptor occurs sequentially, with Site Ibinding first. Site II then engages a second GH receptor, resulting inreceptor dimerization and activation of the intracellular signalingpathways that lead to cellular responses to the hormone. An hGH muteinin which a G120R substitution has been introduced into site II is ableto bind a single hGH receptor, but is unable to dimerize two receptors.The mutein acts as an hGH antagonist in vitro, presumably by occupyingreceptor sites without activating intracellular signaling pathways (Fuh,G., et al., Science 256:1677-1680 (1992)).

Recombinant hGH is used as a therapeutic and has been approved for thetreatment of a number of indications. hGH deficiency leads to dwarfism,for example, which has been successfully treated for more than a decadeby exogenous administration of the hormone. In addition to hGHdeficiency, hGH has also been approved for the treatment of renalfailure (in children), Turner's Syndrome, and cachexia in AIDS patients.Recently, the Food and Drug Administration (FDA) has approved hGH forthe treatment of non-GH-dependent short stature. hGH is also currentlyunder investigation for the treatment of aging, frailty in the elderly,short bowel syndrome, and congestive heart failure. Target populationsfor hGH treatment include children with idiopathic short stature (ISS)and adults with GHD-like symptoms.

Recombinant hGH is currently sold as a daily injectable product, withfive major products currently on the market: Humatrope™ (Eli Lilly &Co.), Nutropin™ (Genentech), Norditropin™ (Novo-Nordisk), Genotropin™(Pfizer) and Saizen/Serostim™ (Serono). A significant challenge to usinggrowth hormone as a therapeutic, however, is that the protein has ashort in vivo half-life and, therefore, it must be administered by dailysubcutaneous injection for maximum effectiveness (MacGillivray, et al.,J. Clin. Endocrinol. Metab. 81: 1806-1809 (1996)). Considerable effortis focused on means to improve the administration of hGH agonists andantagonists, by lowering the cost of production, making administrationeasier for the patient, improving efficacy and safety profile, andcreating other properties that would provide a competitive advantage.For example, Genentech and Alkermes formerly marketed Nutropin Depot™, adepot formulation of hGH, for pediatric growth hormone deficiency. Whilethe depot permits less frequent administration (once every 2-3 weeksrather than once daily), it is also associated with undesirable sideeffects, such as decreased bioavailability and pain at the injectionsite and was withdrawn from the market in 2004. Another product,Pegvisomant™ (Pfizer), has also recently been approved by the FDA.Pegvisomant™ is a genetically-engineered analogue of hGH that functionsas a highly selective growth hormone receptor antagonist indicated forthe treatment of acromegaly (van der Lely, et al., The Lancet 358:1754-1759 (2001). Although several of the amino acid side chain residuesin Pegvisomant™ are derivatized with polyethylene glycol (PEG) polymers,the product is still administered once-daily, indicating that thepharmaceutical properties are not optimal. In addition to PEGylation anddepot formulations, other administration routes, including inhaled andoral dosage forms of hGH, are under early-stage pre-clinical andclinical development and none have yet received approval from the FDA.Accordingly, there is a need for a polypeptide that exhibits growthhormone activity but that also provides a longer serum half-life and,therefore, more optimal therapeutic levels of hGH and an increasedtherapeutic half-life.

Covalent attachment of the hydrophilic polymer poly(ethylene glycol),abbreviated PEG, is a method of increasing water solubility,bioavailability, increasing serum half-life, increasing therapeutichalf-life, modulating immunogenicity, modulating biological activity, orextending the circulation time of many biologically active molecules,including proteins, peptides, and particularly hydrophobic molecules.PEG has been used extensively in pharmaceuticals, on artificialimplants, and in other applications where biocompatibility, lack oftoxicity, and lack of immunogenicity are of importance. In order tomaximize the desired properties of PEG, the total molecular weight andhydration state of the PEG polymer or polymers attached to thebiologically active molecule must be sufficiently high to impart theadvantageous characteristics typically associated with PEG polymerattachment, such as increased water solubility and circulating halflife, while not adversely impacting the bioactivity of the parentmolecule.

PEG derivatives are frequently linked to biologically active moleculesthrough reactive chemical functionalities, such as lysine, cysteine andhistidine residues, the N-terminus and carbohydrate moieties. Proteinsand other molecules often have a limited number of reactive sitesavailable for polymer attachment. Often, the sites most suitable formodification via polymer attachment play a significant role in receptorbinding, and are necessary for retention of the biological activity ofthe molecule. As a result, indiscriminate attachment of polymer chainsto such reactive sites on a biologically active molecule often leads toa significant reduction or even total loss of biological activity of thepolymer-modified molecule. R. Clark et al., (1996), J. Biol. Chem.,271:21969-21977. To form conjugates having sufficient polymer molecularweight for imparting the desired advantages to a target molecule, priorart approaches have typically involved random attachment of numerouspolymer arms to the molecule, thereby increasing the risk of a reductionor even total loss in bioactivity of the parent molecule.

Reactive sites that form the loci for attachment of PEG derivatives toproteins are dictated by the protein's structure. Proteins, includingenzymes, are composed of various sequences of alpha-amino acids, whichhave the general structure H₂N—CHR—COOH. The alpha amino moiety (H₂N—)of one amino acid joins to the carboxyl moiety (—COOH) of an adjacentamino acid to form amide linkages, which can be represented as—(NH—CHR—CO)_(n)—, where the subscript “n” can equal hundreds orthousands. The fragment represented by R can contain reactive sites forprotein biological activity and for attachment of PEG derivatives.

For example, in the case of the amino acid lysine, there exists an —NH₂moiety in the epsilon position as well as in the alpha position. Theepsilon —NH₂ is free for reaction under conditions of basic pH. Much ofthe art in the field of protein derivatization with PEG has beendirected to developing PEG derivatives for attachment to the epsilon—NH₂moiety of lysine residues present in proteins. “Polyethylene Glycol andDerivatives for Advanced PEGylation”, Nektar Molecular EngineeringCatalog, 2003, pp. 1-17. These PEG derivatives all have the commonlimitation, however, that they cannot be installed selectively among theoften numerous lysine residues present on the surfaces of proteins. Thiscan be a significant limitation in instances where a lysine residue isimportant to protein activity, existing in an enzyme active site forexample, or in cases where a lysine residue plays a role in mediatingthe interaction of the protein with other biological molecules, as inthe case of receptor binding sites.

A second and equally important complication of existing methods forprotein PEGylation is that the PEG derivatives can undergo undesiredside reactions with residues other than those desired. Histidinecontains a reactive imino moiety, represented structurally as —N(H)—,but many chemically reactive species that react with epsilon —NH₂ canalso react with —N(H)—. Similarly, the side chain of the amino acidcysteine bears a free sulfhydryl group, represented structurally as —SH.In some instances, the PEG derivatives directed at the epsilon —NH₂group of lysine also react with cysteine, histidine or other residues.This can create complex, heterogeneous mixtures of PEG-derivatizedbioactive molecules and risks destroying the activity of the bioactivemolecule being targeted. It would be desirable to develop PEGderivatives that permit a chemical functional group to be introduced ata single site within the protein that would then enable the selectivecoupling of one or more PEG polymers to the bioactive molecule atspecific sites on the protein surface that are both well-defined andpredictable.

In addition to lysine residues, considerable effort in the art has beendirected toward the development of activated PEG reagents that targetother amino acid side chains, including cysteine, histidine and theN-terminus. See, e.g., U.S. Pat. No. 6,610,281 which is incorporated byreference herein, and “Polyethylene Glycol and Derivatives for AdvancedPEGylation”, Nektar Molecular Engineering Catalog, 2003, pp. 1-17. Acysteine residue can be introduced site-selectively into the structureof proteins using site-directed mutagenesis and other techniques knownin the art, and the resulting free sulfhydryl moiety can be reacted withPEG derivatives that bear thiol-reactive functional groups. Thisapproach is complicated, however, in that the introduction of a freesulfhydryl group can complicate the expression, folding and stability ofthe resulting protein. Thus, it would be desirable to have a means tointroduce a chemical functional group into bioactive molecules thatenables the selective coupling of one or more PEG polymers to theprotein while simultaneously being compatible with (i.e., not engagingin undesired side reactions with) sulfhydryls and other chemicalfunctional groups typically found in proteins.

As can be seen from a sampling of the art, many of these derivativesthat have been developed for attachment to the side chains of proteins,in particular, the —NH₂ moiety on the lysine amino acid side chain andthe —SH moiety on the cysteine side chain, have proven problematic intheir synthesis and use. Some form unstable linkages with the proteinthat are subject to hydrolysis and therefore decompose, degrade, or areotherwise unstable in aqueous environments, such as in the bloodstream.Some form more stable linkages, but are subject to hydrolysis before thelinkage is formed, which means that the reactive group on the PEGderivative may be inactivated before the protein can be attached. Someare somewhat toxic and are therefore less suitable for use in vivo. Someare too slow to react to be practically useful. Some result in a loss ofprotein activity by attaching to sites responsible for the protein'sactivity. Some are not specific in the sites to which they will attach,which can also result in a loss of desirable activity and in a lack ofreproducibility of results. In order to overcome the challengesassociated with modifying proteins with poly(ethylene glycol) moieties,PEG derivatives have been developed that are more stable (e.g., U.S.Pat. No. 6,602,498, which is incorporated by reference herein) or thatreact selectively with thiol moieties on molecules and surfaces (e.g.,U.S. Pat. No. 6,610,281, which is incorporated by reference herein).There is clearly a need in the art for PEG derivatives that arechemically inert in physiological environments until called upon toreact selectively to form stable chemical bonds.

Recently, an entirely new technology in the protein sciences has beenreported, which promises to overcome many of the limitations associatedwith site-specific modifications of proteins. Specifically, newcomponents have been added to the protein biosynthetic machinery of theprokaryote Escherichia coli (E. coli) (e.g., L. Wang, et al., (2001),Science 292:498-500) and the eukaryote Sacchromyces cerevisiae (S.cerevisiae) (e.g., J. Chin et al., Science 301:964-7 (2003)), which hasenabled the incorporation of non-genetically encoded amino acids toproteins in vivo. A number of new amino acids with novel chemical,physical or biological properties, including photoaffinity labels andphotoisomerizable amino acids, photocrosslinking amino acids (see, e.g.,Chin, J. W., et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99:11020-11024;and, Chin, J. W., et al., (2002) J. Am. Chem. Soc. 124:9026-9027), ketoamino acids, heavy atom containing amino acids, and glycosylated aminoacids have been incorporated efficiently and with high fidelity intoproteins in E. coli and in yeast in response to the amber codon, TAG,using this methodology. See, e.g., J. W. Chin et al., (2002), Journal ofthe American Chemical Society 124:9026-9027; J. W. Chin, & P. G.Schultz, (2002), Chem Bio Chem 3(11):1135-1137; J. W. Chin, et al.,(2002), PNAS United States of America 99:11020-11024; and, L. Wang, & P.G. Schultz, (2002), Chem. Comm. 1:1-11. All references are incorporatedby reference in their entirety. These studies have demonstrated that itis possible to selectively and routinely introduce chemical functionalgroups, such as ketone groups, alkyne groups and azide moieties, thatare not found in proteins, that are chemically inert to all of thefunctional groups found in the 20 common, genetically-encoded aminoacids and that may be used to react efficiently and selectively to formstable covalent linkages.

The ability to incorporate non-genetically encoded amino acids intoproteins permits the introduction of chemical functional groups thatcould provide valuable alternatives to the naturally-occurringfunctional groups, such as the epsilon —NH₂ of lysine, the sulfhydryl—SH of cysteine, the imino group of histidine, etc. Certain chemicalfunctional groups are known to be inert to the functional groups foundin the 20 common, genetically-encoded amino acids but react cleanly andefficiently to form stable linkages. Azide and acetylene groups, forexample, are known in the art to undergo a Huisgen [3+2]cycloadditionreaction in aqueous conditions in the presence of a catalytic amount ofcopper. See, e.g., Tornoe, et al., (2002) J. Org. Chem. 67:3057-3064;and, Rostovtsev, et al., (2002) Angew. Chem. Int. Ed. 41:2596-2599. Byintroducing an azide moiety into a protein structure, for example, oneis able to incorporate a functional group that is chemically inert toamines, sulfhydryls, carboxylic acids, hydroxyl groups found inproteins, but that also reacts smoothly and efficiently with anacetylene moiety to form a cycloaddition product. Importantly, in theabsence of the acetylene moiety, the azide remains chemically inert andunreactive in the presence of other protein side chains and underphysiological conditions.

The present invention addresses, among other things, problems associatedwith the activity and production of GH polypeptides, and also addressesthe production of a hGH polypeptide with improved biological orpharmacological properties, such as improved therapeutic half-life.

BRIEF SUMMARY OF THE INVENTION

This invention provides GH supergene family members, including GH, e.g.,hGH polypeptides, comprising one or more non-naturally encoded aminoacids.

In some embodiments, the GH, e.g., hGH polypeptide comprises one or morepost-translational modifications. In some embodiments, the GH, e.g., hGHpolypeptide is linked to a linker, polymer, or biologically activemolecule. In some embodiments, the GH, e.g., hGH polypeptide is linkedto a bifunctional polymer, bifunctional linker, or at least oneadditional GH, e.g., hGH polypeptide.

In some embodiments, the non-naturally encoded amino acid is linked to awater soluble polymer. In some embodiments, the water soluble polymercomprises a poly(ethylene glycol) moiety. In some embodiments, thenon-naturally encoded amino acid is linked to the water soluble polymerwith a linker or is bonded to the water soluble polymer. In someembodiments, the poly(ethylene glycol) molecule is a bifunctionalpolymer. In some embodiments, the bifunctional polymer is linked to asecond polypeptide. In some embodiments, the second polypeptide is a GH,e.g., hGH polypeptide.

In some embodiments, the GH, e.g., hGH polypeptide comprises at leasttwo amino acids linked to a water soluble polymer comprising apoly(ethylene glycol) moiety. In some embodiments, at least one aminoacid is a non-naturally encoded amino acid.

Regions of GH, e.g., hGH can be illustrated as follows, wherein theamino acid positions in hGH are indicated in the middle row:

In some embodiments, one or more non-naturally encoded amino acids areincorporated at any position in one or more of the following regionscorresponding to secondary structures in hGH as follows: 1-5(N-terminus), 6-33 (A helix), 34-74 (region between A helix and B helix,the A-B loop), 75-96 (B helix), 97-105 (region between B helix and Chelix, the B-C loop), 106-129 (C helix), 130-153 (region between C helixand D helix, the C-D loop), 154-183 (D helix), 184-191 (C-terminus) fromSEQ ID NO: 2 or the corresponding amino acids of SEQ ID NO: 1 or 3. Inother embodiments, the non-naturally encoded amino acid is substitutedat a position selected from the group consisting of residues 1-5, 32-46,97-105, 132-149, and 184-191 from hGH SEQ ID NO: 2 or the correspondingamino acids of SEQ ID NO: 1 or 3. In some embodiments, one or morenon-naturally encoded amino acids are incorporated in one or more of thefollowing positions in GH, e.g., hGH: before position 1 (i.e. at theN-terminus), 1, 2, 3, 4, 5, 8, 9, 11, 12, 15, 16, 19, 22, 29, 30, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 52,55, 57, 59, 65, 66, 69, 70, 71, 74, 88, 91, 92, 94, 95, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 111, 112, 113, 115, 116,119, 120, 122, 123, 126, 127, 129, 130, 131, 132, 133, 134, 135, 136,137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 158, 159, 161, 168, 172, 183, 184, 185,186, 187, 188, 189, 190, 191, 192 (i.e., at the carboxyl terminus of theprotein) (SEQ ID NO: 2 or the corresponding amino acids of SEQ ID NO: 1or 3).

In some embodiments, one or more non-naturally encoded amino acids aresubstituted at one or more of the following positions: 29, 30, 33, 34,35, 37, 39, 40, 49, 57, 59, 66, 69, 70, 71, 74, 88, 91, 92, 94, 95, 98,99, 101, 103, 107, 108, 111, 122, 126, 129, 130, 131, 133, 134, 135,136, 137, 139, 140, 141, 142, 143, 145, 147, 154, 155, 156, 159, 183,186, and 187 (SEQ ID NO: 2 or the corresponding amino acids of SEQ IDNO: 1 or 3).

In some embodiments, one or more non-naturally encoded amino acids aresubstituted at one or more of the following positions: 29, 33, 35, 37,39, 49, 57, 69, 70, 71, 74, 88, 91, 92, 94, 95, 98, 99, 101, 103, 107,108, 111, 129, 130, 131, 133, 134, 135, 136, 137, 139, 140, 141, 142,143, 145, 147, 154, 155, 156, 186, and 187 (SEQ ID NO: 2 or thecorresponding amino acids of SEQ ID NO: 1 or 3).

In some embodiments, one or more non-naturally encoded amino acids aresubstituted at one or more of the following positions: 35, 88, 91, 92,94, 95, 99, 101, 103, 111, 131, 133, 134, 135, 136, 139, 140, 143, 145,and 155 (SEQ ID NO: 2 or the corresponding amino acids of SEQ ID NO: 1or 3).

In some embodiments, one or more non-naturally encoded amino acids aresubstituted at one or more of the following positions: 30, 74, 103 (SEQID NO: 2 or the corresponding amino acids of SEQ ID NO: 1 or 3). In someembodiments, one or more non-naturally encoded amino acids aresubstituted at one or more of the following positions: 35, 92, 143, 145(SEQ ID NO: 2 or the corresponding amino acids of SEQ ID NO: 1 or 3).

In some embodiments, the non-naturally occurring amino acid at one ormore of these positions is linked to a water soluble polymer, includingbut not limited to, positions: before position 1 (i.e. at theN-terminus), 1, 2, 3, 4, 5, 8, 9, 11, 12, 15, 16, 19, 22, 29, 30, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 52,55, 57, 59, 65, 66, 69, 70, 71, 74, 88, 91, 92, 94, 95, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 111, 112, 113, 115, 116,119, 120, 122, 123, 126, 127, 129, 130, 131, 132, 133, 134, 135, 136,137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 158, 159, 161, 168, 172, 183, 184, 185,186, 187, 188, 189, 190, 191, 192 (i.e., at the carboxyl terminus of theprotein) (SEQ ID NO: 2 or the corresponding amino acids of SEQ ID NO: 1or 3). In some embodiments, the non-naturally occurring amino acid atone or more of these positions is linked to a water soluble polymer: 30,35, 74, 92, 103, 143, 145 (SEQ ID NO: 2 or the corresponding amino acidsof SEQ ID NO: 1 or 3). In some embodiments, the non-naturally occurringamino acid at one or more of these positions is linked to a watersoluble polymer: 35, 92, 143, 145 (SEQ ID NO: 2 or the correspondingamino acids of SEQ ID NO: 1 or 3).

Human GH antagonists include, but are not limited to, those withsubstitutions at: 1, 2, 3, 4, 5, 8, 9, 11, 12, 15, 16, 19, 22, 103, 109,112, 113, 115, 116, 119, 120, 123, and 127 or an addition at position 1(i.e., at the N-terminus), or any combination thereof (SEQ ID. NO:2, orthe corresponding amino acid in SEQ ID NO: 1, 3, or any other GHsequence).

In some embodiments, the GH, e.g., hGH polypeptide comprises asubstitution, addition or deletion that modulates affinity of the GH,e.g., hGH polypeptide for a GH, e.g., hGH polypeptide receptor whencompared with the affinity of the corresponding GH, e.g., hGH withoutthe substitution, addition, or deletion. In some embodiments, the GH,e.g., hGH polypeptide comprises a substitution, addition, or deletionthat increases the stability of the GH, e.g., hGH polypeptide whencompared with the stability of the corresponding GH, e.g., hGH withoutthe substitution, addition, or deletion. In some embodiments, the GH,e.g., hGH polypeptide comprises an amino acid substitution selected fromthe group consisting of F10A, F10H, F10I; M14W, M14Q, M14G; H18D; H21N;G120A; R167N; D171S; E174S; F176Y, I179T or any combination thereof inhGH SEQ ID NO: 2. In some embodiments, the GH, e.g., hGH polypeptidecomprises a substitution, addition, or deletion that modulates theimmunogenicity of the GH, e.g., hGH polypeptide when compared with theimmunogenicity of the corresponding GH, e.g., hGH without thesubstitution, addition, or deletion. In some embodiments, the GH, e.g.,hGH polypeptide comprises a substitution, addition, or deletion thatmodulates serum half-life or circulation time of the GH, e.g., hGHpolypeptide when compared with the serum half-life or circulation timeof the corresponding GH, e.g., hGH without the substitution, addition,or deletion.

In some embodiments, the GH, e.g., hGH polypeptide comprises asubstitution, addition, or deletion that increases the aqueoussolubility of the GH, e.g., hGH polypeptide when compared to aqueoussolubility of the corresponding GH, e.g., hGH without the substitution,addition, or deletion. In some embodiments, the GH, e.g., hGHpolypeptide comprises a substitution, addition, or deletion thatincreases the solubility of the GH, e.g., hGH polypeptide produced in ahost cell when compared to the solubility of the corresponding GH, e.g.,hGH without the substitution, addition, or deletion. In someembodiments, the GH, e.g., hGH polypeptide comprises a substitution,addition, or deletion that increases the expression of the GH, e.g., hGHpolypeptide in a host cell or increases synthesis in vitro when comparedto the expression or synthesis of the corresponding GH, e.g., hGHwithout the substitution, addition, or deletion. In some embodiments,the hGH polypeptide comprises an amino acid substitution G120A. The hGHpolypeptide comprising this substitution retains agonist activity andretains or improves expression levels in a host cell. In someembodiments, the GH, e.g., hGH polypeptide comprises a substitution,addition, or deletion that increases protease resistance of the GH,e.g., hGH polypeptide when compared to the protease resistance of thecorresponding GH, e.g., hGH without the substitution, addition, ordeletion.

In some embodiments the amino acid substitutions in the GH, e.g., hGHpolypeptide may be with naturally occurring or non-naturally occurringamino acids, provided that at least one substitution is with anon-naturally encoded amino acid.

In some embodiments, the non-naturally encoded amino acid comprises acarbonyl group, an acetyl group, an aminooxy group, a hydrazine group, ahydrazide group, a semicarbazide group, an azide group, or an alkynegroup.

In some embodiments, the non-naturally encoded amino acid comprises acarbonyl group. In some embodiments, the non-naturally encoded aminoacid has the structure:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl; R₂ is H, an alkyl, aryl, substituted alkyl, andsubstituted aryl; and R₃ is H, an amino acid, a polypeptide, or an aminoterminus modification group, and R₄ is H, an amino acid, a polypeptide,or a carboxy terminus modification group.

In some embodiments, the non-naturally encoded amino acid comprises anaminooxy group. In some embodiments, the non-naturally encoded aminoacid comprises a hydrazide group. In some embodiments, the non-naturallyencoded amino acid comprises a hydrazine group. In some embodiments, thenon-naturally encoded amino acid residue comprises a semicarbazidegroup.

In some embodiments, the non-naturally encoded amino acid residuecomprises an azide group. In some embodiments, the non-naturally encodedamino acid has the structure:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, substitutedaryl or not present; X is O, N, S or not present; m is 0-10; R₂ is H, anamino acid, a polypeptide, or an amino terminus modification group, andR₃ is H, an amino acid, a polypeptide, or a carboxy terminusmodification group.

In some embodiments, the non-naturally encoded amino acid comprises analkyne group. In some embodiments, the non-naturally encoded amino acidhas the structure:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl; X is O, N, S or not present; m is 0-10, R₂ is H, anamino acid, a polypeptide, or an amino terminus modification group, andR₃ is H, an amino acid, a polypeptide, or a carboxy terminusmodification group.

In some embodiments, the polypeptide is a GH, e.g., hGH polypeptideagonist, partial agonist, antagonist, partial antagonist, or inverseagonist. In some embodiments, the GH, e.g., hGH polypeptide agonist,partial agonist, antagonist, partial antagonist, or inverse agonistcomprises a non-naturally encoded amino acid linked to a water solublepolymer. In some embodiments, the water soluble polymer comprises apoly(ethylene glycol) moiety. In some embodiments, the GH, e.g., hGHpolypeptide agonist, partial agonist, antagonist, partial antagonist, orinverse agonist comprises a non-naturally encoded amino acid and one ormore post-translational modification, linker, polymer, or biologicallyactive molecule. In some embodiments, the non-naturally encoded aminoacid linked to a water soluble polymer is present within the Site IIregion (the region of the protein encompassing the AC helical-bundleface, amino terminal region of helix A and a portion of helix C) of theGH, e.g., hGH polypeptide. In some embodiments, the GH, e.g., hGHpolypeptide comprising a non-naturally encoded amino acid linked to awater soluble polymer prevents dimerization of the GH, e.g., hGHpolypeptide receptor by preventing the GH, e.g., hGH polypeptideantagonist from binding to a second GH, e.g., hGH polypeptide receptormolecule. In some embodiments, an amino acid other than glycine issubstituted for G120 in SEQ ID NO: 2 (hGH). In some embodiments,arginine is substituted for G120 in SEQ ID NO: 2. In some embodiments, anon-naturally encoded amino acid is substituted for G120 in SEQ ID NO:2. In some embodiments, the non-naturally encoded amino acid linked to awater soluble polymer is present within the receptor binding region ofthe GH, e.g., hGH polypeptide or interferes with the receptor binding ofthe GH, e.g., hGH polypeptide.

The present invention also provides isolated nucleic acids comprising apolynucleotide that hybridizes under stringent conditions to SEQ ID NO:21 or 22 wherein the polynucleotide comprises at least one selectorcodon. In some embodiments, the selector codon is selected from thegroup consisting of an amber codon, ochre codon, opal codon, a uniquecodon, a rare codon, a five-base codon, and a four-base codon.

The present invention also provides methods of making a GH, e.g., hGHpolypeptide linked to a water soluble polymer. In some embodiments, themethod comprises contacting an isolated GH, e.g., hGH polypeptidecomprising a non-naturally encoded amino acid with a water solublepolymer comprising a moiety that reacts with the non-naturally encodedamino acid. In some embodiments, the non-naturally encoded amino acidincorporated into the GH, e.g., hGH polypeptide is reactive toward awater soluble polymer that is otherwise unreactive toward any of the 20common amino acids. In some embodiments, the non-naturally encoded aminoacid incorporated into the GH, e.g., hGH polypeptide is reactive towarda linker, polymer, or biologically active molecule that is otherwiseunreactive toward any of the 20 common amino acids.

In some embodiments, the GH, e.g., hGH polypeptide linked to the watersoluble polymer is made by reacting a GH, e.g., hGH polypeptidecomprising a carbonyl-containing amino acid with a poly(ethylene glycol)molecule comprising an aminooxy, hydrazine, hydrazide or semicarbazidegroup. In some embodiments, the aminooxy, hydrazine, hydrazide orsemicarbazide group is linked to the poly(ethylene glycol) moleculethrough an amide linkage.

In some embodiments, the GH, e.g., hGH polypeptide linked to the watersoluble polymer is made by reacting a poly(ethylene glycol) moleculecomprising a carbonyl group with a polypeptide comprising anon-naturally encoded amino acid that comprises an aminooxy, hydrazine,hydrazide or semicarbazide group.

In some embodiments, the GH, e.g., hGH polypeptide linked to the watersoluble polymer is made by reacting a GH, e.g., hGH polypeptidecomprising an alkyne-containing amino acid with a poly(ethylene glycol)molecule comprising an azide moiety. In some embodiments, the azide oralkyne group is linked to the poly(ethylene glycol) molecule through anamide linkage.

In some embodiments, the GH, e.g., hGH polypeptide linked to the watersoluble polymer is made by reacting a GH, e.g., hGH polypeptidecomprising an azide-containing amino acid with a poly(ethylene glycol)molecule comprising an alkyne moiety. In some embodiments, the azide oralkyne group is linked to the poly(ethylene glycol) molecule through anamide linkage.

In some embodiments, the poly(ethylene glycol) molecule has a molecularweight of between about 0.1 kDa and about 100 kDa. In some embodiments,the poly(ethylene glycol) molecule has a molecular weight of between 0.1kDa and 50 kDa.

In some embodiments, the poly(ethylene glycol) molecule is a branchedpolymer. In some embodiments, each branch of the poly(ethylene glycol)branched polymer has a molecular weight of between 1 kDa and 100 kDa, orbetween 1 kDa and 50 kDa.

In some embodiments, the water soluble polymer linked to the GH, e.g.,hGH polypeptide comprises a polyalkylene glycol moiety. In someembodiments, the non-naturally encoded amino acid residue incorporatedinto the GH, e.g., hGH polypeptide comprises a carbonyl group, anaminooxy group, a hydrazide group, a hydrazine, a semicarbazide group,an azide group, or an alkyne group. In some embodiments, thenon-naturally encoded amino acid residue incorporated into the GH, e.g.,hGH polypeptide comprises a carbonyl moiety and the water solublepolymer comprises an aminooxy, hydrazide, hydrazine, or semicarbazidemoiety. In some embodiments, the non-naturally encoded amino acidresidue incorporated into the GH, e.g., hGH polypeptide comprises analkyne moiety and the water soluble polymer comprises an azide moiety.In some embodiments, the non-naturally encoded amino acid residueincorporated into the GH, e.g., hGH polypeptide comprises an azidemoiety and the water soluble polymer comprises an alkyne moiety.

The present invention also provides compositions comprising a GH, e.g.,hGH polypeptide comprising a non-naturally encoded amino acid and apharmaceutically acceptable carrier. In some embodiments, thenon-naturally encoded amino acid is linked to a water soluble polymer.

The present invention also provides cells comprising a polynucleotideencoding the GH, e.g., hGH polypeptide comprising a selector codon. Insome embodiments, the cells comprise an orthogonal RNA synthetase and/oran orthogonal tRNA for substituting a non-naturally encoded amino acidinto the GH, e.g., hGH polypeptide.

The present invention also provides methods of making a GH, e.g., hGHpolypeptide comprising a non-naturally encoded amino acid. In someembodiments, the methods comprise culturing cells comprising apolynucleotide or polynucleotides encoding a GH, e.g., hGH polypeptide,an orthogonal RNA synthetase and/or an orthogonal tRNA under conditionsto permit expression of the GH, e.g., hGH polypeptide; and purifying theGH, e.g., hGH polypeptide from the cells and/or culture medium.

The present invention also provides methods of increasing therapeutichalf-life, serum half-life or circulation time of GH, e.g., hGHpolypeptides. The present invention also provides methods of modulatingimmunogenicity of GH, e.g., hGH polypeptides. In some embodiments, themethods comprise substituting a non-naturally encoded amino acid for anyone or more amino acids in naturally occurring GH, e.g., hGHpolypeptides and/or linking the GH, e.g., hGH polypeptide to a linker, apolymer, a water soluble polymer, or a biologically active molecule.

The present invention also provides methods of treating a patient inneed of such treatment with an effective amount of a GH, e.g., hGHmolecule of the present invention. In some embodiments, the methodscomprise administering to the patient a therapeutically-effective amountof a pharmaceutical composition comprising a GH, e.g., hGH polypeptidecomprising a non-naturally-encoded amino acid and a pharmaceuticallyacceptable carrier. In some embodiments, the non-naturally encoded aminoacid is linked to a water soluble polymer.

The present invention also provides GH, e.g., hGH polypeptidescomprising a sequence shown in SEQ ID NO: 1, 2, 3, or any other GHpolypeptide sequence, except that at least one amino acid is substitutedby a non-naturally encoded amino acid. In some embodiments, thenon-naturally encoded amino acid is linked to a water soluble polymer.In some embodiments, the water soluble polymer comprises a poly(ethyleneglycol) moiety. In some embodiments, the non-naturally encoded aminoacid comprises a carbonyl group, an aminooxy group, a hydrazide group, ahydrazine group, a semicarbazide group, an azide group, or an alkynegroup. In some embodiments, the non-naturally encoded amino acid issubstituted at a position selected from the group consisting of residues1-5, 82-90, 117-134, and 169-176 from SEQ ID NO: 3 (hGH).

The present invention also provides pharmaceutical compositionscomprising a pharmaceutically acceptable carrier and a GH, e.g., hGHpolypeptide comprising the sequence shown in SEQ ID NO: 1, 2, 3, or anyother GH polypeptide sequence, wherein at least one amino acid issubstituted by a non-naturally encoded amino acid. In some embodiments,the non-naturally encoded amino acid comprises a saccharide moiety. Insome embodiments, the water soluble polymer is linked to the polypeptidevia a saccharide moiety. In some embodiments, a linker, polymer, orbiologically active molecule is linked to the GH, e.g., hGH polypeptidevia a saccharide moiety.

The present invention also provides a GH, e.g., hGH polypeptidecomprising a water soluble polymer linked by a covalent bond to the GH,e.g., hGH polypeptide at a single amino acid. In some embodiments, thewater soluble polymer comprises a poly(ethylene glycol) moiety. In someembodiments, the amino acid covalently linked to the water solublepolymer is a non-naturally encoded amino acid present in thepolypeptide. In some embodiments the non-naturally encoded amino acid issubstituted at position 35, 92, 143, or 145 of SEQ ID NO 2.

The present invention provides a GH, e.g., hGH polypeptide comprising atleast one linker, polymer, or biologically active molecule, wherein saidlinker, polymer, or biologically active molecule is attached to thepolypeptide through a functional group of a non-naturally encoded aminoacid ribosomally incorporated into the polypeptide. In some embodiments,the polypeptide is monoPEGylated. The present invention also provides aGH, e.g., hGH polypeptide comprising a linker, polymer, or biologicallyactive molecule that is attached to one or more non-naturally encodedamino acid wherein said non-naturally encoded amino acid is ribosomallyincorporated into the polypeptide at pre-selected sites.

In another embodiment, conjugation of the hGH polypeptide comprising oneor more non-naturally occurring amino acids to another molecule,including but not limited to PEG, provides substantially purified hGHdue to the unique chemical reaction utilized for conjugation to thenon-natural amino acid. Conjugation of hGH comprising one or morenon-naturally encoded amino acids to another molecule, such as PEG, maybe performed with other purification techniques performed prior to orfollowing the conjugation step to provide substantially pure hGH.

The present invention further provides a hormone composition containinga growth hormone (GH) linked to at least one water-soluble polymer by acovalent bond, where the covalent bond is an oxime bond. In someembodiments, the GH is a human growth hormone (hGH), such as a sequencethat is at least about 80% identical to SEQ ID NO: 2; in someembodiments the sequence is the sequence of SEQ ID NO: 2. The GH caninclude one or more non-naturally encoded amino acids (NEAAs), such as aNEAA that includes a carbonyl group, e.g., a ketone, such as an NEAAthat is para-acetylphenylalanine. In some embodiments the oxime bond isbetween the NEAA and the water-soluble polymer. The GH can besubstituted with a para-acetylphenylalanine at a position correspondingto position 35 of SEQ ID NO: 2. In some embodiments, the water-solublepolymer includes one or more polyethylene glycol (PEG) molecules. ThePEG can be linear, e.g., a linear PEG of MW of about 0.1 and about 100kDa, or about 1 and about 60 kDa, or about 20 and about 40 kDa, or about30 kDa. In some embodiments, the PEG is a branched PEG, e.g., a branchedPEG that has a molecular weight between about 1 and about 100 kDa, orabout 30 and about 50 kDa, or about 40 kDa. In some embodiments the GHis linked by a plurality of covalent bonds to a plurality ofwater-soluble polymers, where at least one of the covalent bonds areoxime bonds. In some of these embodiments, the GH is a human growthhormone (GH, e.g., hGH), e.g., a GH, e.g., hGH with a sequence that isat least about 80% identical to SEQ ID NO: 2; in some embodiments thesequence is that of SEQ ID NO: 2. In some embodiments in which the GH,e.g., hGH, is linked to a plurality of water-soluble polymers, the GHcomprises a plurality of NEAAs.

In certain embodiments, the invention provides a GH composition thatcontains a GH, e.g., hGH that comprises the sequence of SEQ ID NO: 2,where the GH, e.g., hGH is linked via an oxime bond to a 30 kDa linearPEG, and where the oxime bond is formed with a para-acetylphenylalaninesubstituted at a position corresponding to position 35 of SEQ ID NO: 2.

In some embodiments, the invention provides a hormone compositioncontaining a GH, e.g., hGH linked via an oxime bond to at least onelinear PEG, where the GH, e.g., hGH comprises the amino acid sequence ofSEQ ID NO: 2 and contains at least one NEAA substituted at one or morepositions selected from the group consisting of residues 1-5, 6-33,34-74, 75-96, 97-105, 106-129, 130-153, 154-183, and 184-191. In someembodiments, the NEAA(s) is substituted at one or more positionsselected from the group consisting of residues before position 1 (i.e.at the N-terminus), 1, 2, 3, 4, 5, 8, 9, 11, 12, 15, 16, 19, 22, 29, 30,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,52, 55, 57, 59, 65, 66, 69, 70, 71, 74, 88, 91, 92, 94, 95, 97, 98, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 111, 112, 113, 115,116, 119, 120, 122, 123, 126, 127, 129, 130, 131, 132, 133, 134, 135,136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149,150, 151, 152, 153, 154, 155, 156, 158, 159, 161, 168, 172, 183, 184,185, 186, 187, 188, 189, 190, 191, and 192 (i.e., at the carboxylterminus of the protein). In some embodiments, the NEAA(s) issubstituted at one or more positions selected from the group consistingof residues 35, 92, 131, 134, 143, and 145. In some embodiments, theNEAA(s) is substituted at one or more positions selected from the groupconsisting of residues 30, 35, 74, 92, 103, 143, and 145. In someembodiments, the NEAA(s) is substituted at one or more positionsselected from the group consisting of residues 35, 92, 143, and 145. Insome embodiments, the NEAA is substituted at position 35. At least oneof the NEAA is a para-acetylphenylalanine in some embodiments. In someembodiments, the PEG has a molecular weight between about 0.1 and about100 kDa, or about 1 and about 60 kDa, or about 20 and about 40 kDa, orabout 30 kDa.

In yet other embodiments, the invention provides a method of making aGH, e.g., hGH linked via an oxime bond to a water-soluble polymercomprising contacting a GH, e.g., hGH that comprises a NEAA comprising acarbonyl group with a PEG oxyamine under conditions suitable forformation of an oxime bond. The NEAA can contain a ketone group, e.g., acarbonyl. The NEAA can be para-acetylphenylalanine. In some embodimentscontaining a para-acetylphenylalanine, the para-acetylphenylalanine issubstituted at a position in the GH, e.g., hGH corresponding to aminoacid 35 in SEQ ID NO: 2. In some embodiments, the PEG oxyamine is amonomethoxyPEG (MPEG) oxyamine. In some embodiments, the MPEG oxyamineis linear, e.g., a linear MPEG of about 20-40 kDa, or about 30 kDa. Insome embodiments, the MPEG oxyamine is a linear 30 kDamonomethoxy-PEG-2-aminooxy ethylamine carbamate hydrochloride. In someembodiments, the GH, e.g., hGH comprising an NEAA is made by introducing(i) a nucleic acid encoding a GH, e.g., hGH wherein the nucleic acid hasbeen modified to provide a selector codon for incorporation of the NEAA;and (ii) the NEAA; to an organism whose cellular machinery is capable ofincorporating the NEAA into a protein in response to the selector codonof the nucleic acid of (i). In some embodiments, the reaction conditionsfor forming the oxime bond include mixing the MPEG and GH, e.g., hGH toproduce a MPEG-GH, e.g., hGH mixture with a MPEG:GH, e.g., hGH ratio ofabout 5 to 10, a pH of about 4 to 6; and gentle stirring of the MPEG-GH,e.g., MPEG-hGH mixture for about 10 to 50 hours at room temperature. Insome embodiments, the method further includes purifying the GH, e.g.,hGH, e.g., to at least about 99% pure.

Cellular machinery includes, but is not limited to, an orthogonal tRNAand/or aminoacyl tRNA synthetase.

In still yet further embodiments, the invention provides apharmaceutical composition that contains a hormone compositioncomprising a growth hormone linked by a covalent bond to at least onewater-soluble polymer, wherein the covalent bond is an oxime bond, and apharmaceutically acceptable excipient. In some embodiments, the GH is aGH, e.g., hGH. In some embodiments, the GH comprises a NEAA. In someembodiments, the water-soluble polymer comprises a PEG, such as a linearPEG. In some embodiments the PEG is a linear PEG of about 30 kDa and theGH is an GH, e.g., hGH substituted at a position corresponding to aminoacid 35 of SEQ ID NO: 2 with a para-acetylphenylalanine, and the oximebond is formed between the para-acetylphenylalanine and the PEG.

In some embodiments, the invention provides a method of treatment byadministering to an individual in need of treatment an effective amountof a hormone composition containing a growth hormone (GH) linked bycovalent bond(s) to at least one water-soluble polymer, where thecovalent bond(s) is an oxime bond. In some embodiments, the GH is hGH.In some embodiments, the GH comprises a NEAA. In some embodiments, thewater-soluble polymer comprises a PEG, such as a linear PEG. In someembodiments, the PEG is a linear PEG of about 30 kDa and the GH is hGHsubstituted at a position corresponding to amino acid 35 of SEQ ID NO: 2with a para-acetylphenylalanine, and the oxime bond is formed betweenthe para-acetylphenylalanine and the PEG. In some embodiments, theindividual that is treated is a human. In some embodiments, theindividual that is treated suffers from pediatric growth hormonedeficiency, idiopathic short stature, adult growth hormone deficiency ofchildhood onset, adult growth hormone deficiency of adult onset, orsecondary growth hormone deficiency. In some embodiments, the GH is hGH.In some embodiments, the GH comprises a NEAA. In some embodiments, thewater-soluble polymer comprises a PEG, such as a linear PEG. In someembodiments, the PEG is a linear PEG of about 30 kDa and the GH e.g.,hGH substituted at a position corresponding to amino acid 35 of SEQ IDNO: 2 with a para-acetylphenylalanine, and the oxime bond is formedbetween the para-acetylphenylalanine and the PEG.

In some embodiments, the invention provides a method of treatment byadministering to an individual in need of treatment an effective amountof a hormone composition comprising a growth hormone (GH) linked bycovalent bond(s) to at least one water-soluble polymer, where thewater-soluble polymer is a linear polymer, and where the hormonecomposition is given at a frequency of no more than once per week, onceper two weeks, or once per month. In some embodiments, the polymer is aPEG. In some embodiments, the GH comprises a NEAA. In some embodiments,the polymer is linked to the GH via an oxime bond.

In some embodiments, the invention provides a hormone compositioncomprising a GH, e.g., hGH, wherein the GH, e.g., hGH has an averageserum half-life of at least about 12 hours when administered to a mammalsubcutaneously. In some embodiments, the GH, e.g., hGH comprises a NEAA.In some embodiments, the hormone composition further contains awater-soluble polymer, such as a PEG, e.g., a linear PEG.

In some embodiments, the invention provides a hormone compositioncomprising a GH, e.g., hGH linked to a PEG, wherein the GH, e.g., hGHhas an average serum half-life of at least about 7-fold the serumhalf-life of a composition comprising the GH, e.g., hGH without the PEG,when administered to a mammal subcutaneously.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1—A diagram of the general structure for four helical bundleproteins is shown.

FIG. 2—A diagram of the general structure for the four helical bundleprotein Growth Hormone (GH) is shown.

FIG. 3—A diagram of the general structure for the four helical bundleprotein Erythropoietin (EPO) is shown.

FIG. 4—A diagram of the general structure for the four helical bundleprotein Interferon alpha-2 (IFNα-2) is shown.

FIG. 5—A diagram of the general structure for the four helical bundleprotein Granulocyte Colony Stimulating Factor (G-CSF) is shown.

FIG. 6—A Coomassie blue stained SDS-PAGE is shown demonstrating theexpression of hGH comprising the non-naturally encoded amino acidp-acetyl phenylalanine at each of the following positions: Y35, F92,Y1111, G131, R134, K140, Y143, or K145.

FIG. 7, Panels A and B—A diagram of the biological activity of the hGHcomprising a non-naturally encoded amino acid (Panel B) and wild-typehGH (Panel A) on IM9 cells is shown.

FIG. 8—A Coomassie blue stained SDS-PAGE is shown demonstrating theproduction of hGH comprising a non-naturally encoded amino acid that isPEGylated by covalent linkage of PEG (5, 20 and 30 kDa) to thenon-naturally encoded amino acid.

FIG. 9—A diagram is shown demonstrating the biological activity of thevarious PEGylated forms of hGH comprising a non-naturally encoded aminoacid on IM9 cells.

FIG. 10, Panel A—This figure depicts the primary structure of hGH withthe trypsin cleavage sites indicated and the non-natural amino acidsubstitution, F92pAF, specified with an arrow (Figure modified fromBecker et al. Biotechnol Appl Biochem. (1988) 10(4):326-337). FIG. 10,Panel B—Superimposed tryptic maps are shown of peptides generated from ahGH polypeptide comprising a non-naturally encoded amino acid that isPEGylated (labeled A), peptides generated from a hGH polypeptidecomprising a non-naturally encoded amino acid (labeled B), and peptidesgenerated from WHO rhGH (labeled C). FIG. 10, Panel C—A magnification ofpeak 9 from Panel B is shown.

FIG. 11, Panel A and Panel B show Coomassie blue stained SDS-PAGEanalysis of purified PEG-hGH polypeptides.

FIG. 12—A diagram of the biological activity of a hGH dimer molecule onIM9 cells is shown.

FIG. 13, Panel A—A diagram is shown of the IM-9 assay data measuringphosphorylation of pSTAT5 by hGH antagonist with the G120R substitution.FIG. 13, Panel B—A diagram is shown of the IM-9 assay data measuringphosphorylation of pSTAT5 by a hGH polypeptide with a non-natural aminoacid incorporated at the same position (G120).

FIG. 14—A diagram is shown indicating that a dimer of the hGH antagonistshown in FIG. 13, Panel B also lacks biological activity in the IM-9assay.

FIG. 15—A diagram is shown comparing the serum half-life in rats of hGHpolypeptide comprising a non-naturally encoded amino acid that isPEGylated with hGH polypeptide that is not PEGylated.

FIG. 16—A diagram is shown comparing the serum half-life in rats of hGHpolypeptides comprising a non-naturally encoded amino acid that isPEGylated.

FIG. 17—A diagram is shown comparing the serum half-life in rats of hGHpolypeptides comprising a non-naturally encoded amino acid that isPEGylated. Rats were dosed once with 2.1 mg/kg.

FIG. 18, Panel A—A diagram is shown of the effect on rat body weightgain after administration of a single dose of hGH polypeptidescomprising a non-naturally encoded amino acid that is PEGylated(position 35, 92). FIG. 18, Panel B—A diagram is shown of the effect oncirculating plasma IGF-1 levels after administration of a single dose ofhGH polypeptides comprising a non-naturally encoded amino acid that isPEGylated (position 35, 92). FIG. 18, Panel C—A diagram is shown of theeffect on rat body weight gain after administration of a single dose ofhGH polypeptides comprising a non-naturally encoded amino acid that isPEGylated (position 92, 134, 145, 131, 143). FIG. 18, Panel D—A diagramis shown of the effect on circulating plasma IGF-1 levels afteradministration of a single dose of hGH polypeptides comprising anon-naturally encoded amino acid that is PEGylated (position 92, 134,145, 131, 143). FIG. 18, Panel E—A diagram is shown comparing the serumhalf-life in rats of hGH polypeptides comprising a non-naturally encodedamino acid that is PEGylated (position 92, 134, 145, 131, 143).

FIG. 19—A diagram is shown of the structure of linear, 30 kDamonomethoxy-poly(ethylene glycol)-2-aminooxy ethylamine carbamatehydrochloride.

FIG. 20—A diagram is shown illustrating synthesis of carbamate-linkedoxyamino-derivatized PEG

FIG. 21 presents illustrative, non-limiting examples of PEG-containingreagents that can be used to modify non-natural amino acid polypeptidesto form PEG-containing, oxime-linked non-natural amino acidpolypeptides.

FIG. 22 presents illustrative, non-limiting examples of the synthesis ofPEG-containing reagents that can be used to modify non-natural aminoacid polypeptides to form PEG-containing, oxime-linked non-natural aminoacid polypeptides.

FIG. 23 presents an illustrative, non-limiting example of the synthesisof an amide-based hydroxylamine PEG-containing reagent that can be usedto modify non-natural amino acid polypeptides to form PEG-containing,oxime-linked non-natural amino acid polypeptides.

FIG. 24 presents an illustrative, non-limiting example of the synthesisof a carbamate-based PEG-containing reagent that can be used to modifynon-natural amino acid polypeptides to form PEG-containing, oxime-linkednon-natural amino acid polypeptides.

FIG. 25 presents an illustrative, non-limiting example of the synthesisof a carbamate-based PEG-containing reagent that can be used to modifynon-natural amino acid polypeptides to form PEG-containing, oxime-linkednon-natural amino acid polypeptides.

FIG. 26 presents illustrative, non-limiting examples of the synthesis ofsimple PEG-containing reagents that can be used to modify non-naturalamino acid polypeptides to form PEG-containing, oxime-linked non-naturalamino acid polypeptides.

FIG. 27 presents illustrative, non-limiting examples of branchedPEG-containing reagents that can be used to modify non-natural aminoacid polypeptides to form PEG-containing, oxime-linked non-natural aminoacid polypeptides, and the use of one such reagent to modify acarbonyl-based non-natural amino acid polypeptide.

FIG. 28 presents a graph illustrating IGF-1 plasma concentration inhypophysectomized rats treated weekly with placebo or increasing dose ofPEG-^(a)hGH, or daily with placebo or Genotropin.

FIG. 29 presents a graph illustrating tibial bone length inhypophysectomized rats treated weekly with placebo or PEG-^(a)hGH, ordaily with placebo or Genotropin.

FIG. 30 presents a graph illustrating percent bodyweight change inhyposphysectomized rats treated weekly with placebo or PEG-^(a)hGH, ordaily with placebo or Genotropin.

FIG. 31 presents a graph illustrating plasma concentration versus timefor PEG-^(a)hGH administered subcutaneously on days 0 and 7.

FIG. 32 presents a graph illustrating plasma concentration versus timefor PEG-^(a)(met)hGH administered as a single subcutaneous orintravenous dose.

DEFINITIONS

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, constructs, and reagentsdescribed herein and as such may vary. It is also to be understood thatthe terminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention, which will be limited only by the appended claims.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly indicatesotherwise. Thus, for example, reference to a “hGH” is a reference to oneor more such proteins and includes equivalents thereof known to those ofordinary skill in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devices,and materials similar or equivalent to those described herein can beused in the practice or testing of the invention, the preferred methods,devices and materials are now described.

All publications and patents mentioned herein are incorporated herein byreference for the purpose of describing and disclosing, for example, theconstructs and methodologies that are described in the publications,which might be used in connection with the presently describedinvention. The publications discussed herein are provided solely fortheir disclosure prior to the filing date of the present application.Nothing herein is to be construed as an admission that the inventors arenot entitled to antedate such disclosure by virtue of prior invention orfor any other reason.

The term “substantially purified” refers to a GH, e.g., hGH polypeptidethat may be substantially or essentially free of components thatnormally accompany or interact with the protein as found in itsnaturally occurring environment, i.e. a native cell, or host cell in thecase of recombinantly produced GH, e.g., hGH polypeptides. GH, e.g., hGHpolypeptide that may be substantially free of cellular material includespreparations of protein having less than about 30%, less than about 25%,less than about 20%, less than about 15%, less than about 10%, less thanabout 5%, less than about 4%, less than about 3%, less than about 2%, orless than about 1% (by dry weight) of contaminating protein. When theGH, e.g., hGH polypeptide or variant thereof is recombinantly producedby the host cells, the protein may be present at about 30%, about 25%,about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%,or about 1% or less of the dry weight of the cells. When the GH, e.g.,hGH polypeptide or variant thereof is recombinantly produced by the hostcells, the protein may be present in the culture medium at about 5 g/L,about 4 g/L, about 3 g/L, about 2 g/L, about 1 g/L, about 750 mg/L,about 500 mg/L, about 250 mg/L, about 100 mg/L, about 50 mg/L, about 10mg/L, or about 1 mg/L or less of the dry weight of the cells. Thus,“substantially purified” GH, e.g., hGH polypeptide as produced by themethods of the present invention may have a purity level of at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 55%, at least about 60%, at leastabout 65%, at least about 70%, specifically, a purity level of at leastabout 75%, 80%, 85%, and more specifically, a purity level of at leastabout 90%, a purity level of at least about 95%, a purity level of atleast about 99% or greater as determined by appropriate methods such asSDS/PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.

A “recombinant host cell” or “host cell” refers to a cell that includesan exogenous polynucleotide, regardless of the method used forinsertion, for example, direct uptake, transduction, f-mating, or othermethods known in the art to create recombinant host cells. The exogenouspolynucleotide may be maintained as a nonintegrated vector, for example,a plasmid, or alternatively, may be integrated into the host genome.

As used herein, the term “medium” or “media” includes any culturemedium, solution, solid, semi-solid, or rigid support that may supportor contain any host cell, including bacterial host cells, yeast hostcells, insect host cells, plant host cells, eukaryotic host cells,mammalian host cells, CHO cells, prokaryotic host cells, E. coli, orPseudomonas host cells, and cell contents. Thus, the term may encompassmedium in which the host cell has been grown, e.g., medium into whichthe GH, e.g., hGH polypeptide has been secreted, including medium eitherbefore or after a proliferation step. The term also may encompassbuffers or reagents that contain host cell lysates, such as in the casewhere the GH, e.g., hGH polypeptide is produced intracellularly and thehost cells are lysed or disrupted to release the GH, e.g., hGHpolypeptide.

“Reducing agent,” as used herein with respect to protein refolding, isdefined as any compound or material which maintains sulfhydryl groups inthe reduced state and reduces intra- or intermolecular disulfide bonds.Suitable reducing agents include, but are not limited to, dithiothreitol(DTT), 2-mercaptoethanol, dithioerythritol, cysteine, cysteamine(2-aminoethanethiol), and reduced glutathione. It is readily apparent tothose of ordinary skill in the art that a wide variety of reducingagents are suitable for use in the methods and compositions of thepresent invention.

“Oxidizing agent,” as used hereinwith respect to protein refolding, isdefined as any compound or material which is capable of removing anelectron from a compound being oxidized. Suitable oxidizing agentsinclude, but are not limited to, oxidized glutathione, cystine,cystamine, oxidized dithiothreitol, oxidized erythreitol, and oxygen. Itis readily apparent to those of ordinary skill in the art that a widevariety of oxidizing agents are suitable for use in the methods of thepresent invention.

“Denaturing agent” or “denaturant,” as used herein, is defined as anycompound or material which will cause a reversible unfolding of aprotein. The strength of a denaturing agent or denaturant will bedetermined both by the properties and the concentration of theparticular denaturing agent or denaturant. Suitable denaturing agents ordenaturants may be chaotropes, detergents, organic solvents, watermiscible solvents, phospholipids, or a combination of two or more suchagents. Suitable chaotropes include, but are not limited to, urea,guanidine, and sodium thiocyanate. Useful detergents may include, butare not limited to, strong detergents such as sodium dodecyl sulfate, orpolyoxyethylene ethers (e.g. Tween or Triton detergents), Sarkosyl, mildnon-ionic detergents (e.g., digitonin), mild cationic detergents such asN->2,3-(Dioleyoxy)-propyl-N,N,N-trimethylammonium, mild ionic detergents(e.g. sodium cholate or sodium deoxycholate) or zwitterionic detergentsincluding, but not limited to, sulfobetaines (Zwittergent),3-(3-chlolamidopropyl)dimethylammonio-1-propane sulfate (CHAPS), and3-(3-chlolamidopropyl)dimethylammonio-2-hydroxy-1-propane sulfonate(CHAPSO). Organic, water miscible solvents such as acetonitrile, loweralkanols (especially C₂-C₄ alkanols such as ethanol or isopropanol), orlower alkandiols (especially C₂-C₄ alkandiols such as ethylene-glycol)may be used as denaturants. Phospholipids useful in the presentinvention may be naturally occurring phospholipids such asphosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, andphosphatidylinositol or synthetic phospholipid derivatives or variantssuch as dihexanoylphosphatidylcholine or diheptanoylphosphatidylcholine.

“Refolding,” as used herein describes any process, reaction or methodwhich transforms disulfide bond containing polypeptides from animproperly folded or unfolded state to a native or properly foldedconformation with respect to disulfide bonds.

“Cofolding,” as used herein, refers specifically to refolding processes,reactions, or methods which employ at least two polypeptides whichinteract with each other and result in the transformation of unfolded orimproperly folded polypeptides to native, properly folded polypeptides.

As used herein, “growth hormone” or “GH” shall include thosepolypeptides and proteins that have at least one biological activity ofa growth hormone from any mammalian species including but not limitedto, human (hGH), bovine (bGH), porcine, and from other livestock or farmanimals including but not limited to, chicken, as well as GH analogs, GHisoforms, GH mimetics, GH fragments, hybrid GH proteins, fusionproteins, oligomers and multimers, homologues, glycosylation patternvariants, variants, splice variants, and muteins, thereof, regardless ofthe biological activity of same, and further regardless of the method ofsynthesis or manufacture thereof including, but not limited to,recombinant (whether produced from cDNA, genomic DNA, synthetic DNA orother form of nucleic acid), in vitro, in vivo, by microinjection ofnucleic acid molecules, synthetic, transgenic, and gene activatedmethods.

The term “hGH polypeptide” encompasses hGH polypeptides comprising oneor more amino acid substitutions, additions or deletions. Exemplarysubstitutions include, e.g., substitution of the lysine at position 41or the phenylalanine at position 176 of native hGH. In some cases, thesubstitution may be an isoleucine or arginine residue if thesubstitution is at position 41 or is a tyrosine residue if the positionis 176. Position F10 can be substituted with, e.g., A, H or I. PositionM14 may be substituted with, e.g., W, Q or G. Other exemplarysubstitutions include any substitutions or combinations thereof,including but not limited to:

R167N, D171S, E174S, F176Y, I179T;

R167E, D171S, E174S, F176Y;

F10A, M14W, H18D, H21N;

F10A, M14W, H18D, H21N, R167N, D171S, E174S, F176Y, I179T;

F10A, M14W, H18D, H21N, R167N, D171A, E174S, F176Y, I179T;

F10H, M14G, H18N, H21N;

F10A, M14W, H18D, H21N, R167N, D171A, T175T, I179T; or

F10I, M14Q, H18E, R167N, D171S, I179T. See, e.g., U.S. Pat. No.6,143,523, which is incorporated by reference herein.

Exemplary substitutions in a wide variety of amino acid positions innaturally-occurring hGH have been described, including substitutionsthat increase agonist activity, increase protease resistance, convertthe polypeptide into an antagonist, etc. and are encompassed by the term“hGH polypeptide.”

Agonist GH, e.g., hGH sequences include, e.g., the naturally-occurringhGH sequence comprising the following modifications H18D, H21N, R167N,D171S, E174S, I179T. See, e.g., U.S. Pat. No. 5,849,535, which isincorporated by reference herein. Additional agonist hGH sequencesinclude

H18D, Q22A, F25A, D26A, Q29A, E65A, K168A, E174S;

H18A, Q22A, F25A, D26A, Q29A, E65A, K168A, E174S; or

H18D, Q22A, F25A, D26A, Q29A, E65A, K168A, E174A. See, e.g. U.S. Pat.No. 6,022,711, which is incorporated by reference herein. hGHpolypeptides comprising substitutions at H18A, Q22A, F25A, D26A, Q29A,E65A, K168A, E174A enhance affinity for the hGH receptor at site I. See,e.g. U.S. Pat. No. 5,854,026, which is incorporated by reference herein.hGH sequences with increased resistance to proteases include, but arenot limited to, hGH polypeptides comprising one or more amino acidsubstitutions within the C-D loop. In some embodiments, substitutionsinclude, but are not limited to, R134D, T135P, K140A, and anycombination thereof. See, e.g., Alam et al. (1998) J. Biotechnol.65:183-190.

Human Growth Hormone antagonists include, e.g., those with asubstitution at G120 (e.g., G120R, G120K, G120W, G120Y, G120F, or G120E)and sometimes further including the following substitutions: H18A, Q22A,F25A, D26A, Q29A, E65A, K168A, E174A. See, e.g. U.S. Pat. No. 6,004,931,which is incorporated by reference herein. In some embodiments, hGHantagonists comprise at least one substitution in the regions 106-108 or127-129 that cause GH to act as an antagonist. See, e.g., U.S. Pat. No.6,608,183, which is incorporated by reference herein. In someembodiments, the hGH antagonist comprises a non-naturally encoded aminoacid linked to a water soluble polymer that is present in the Site IIbinding region of the hGH molecule. In some embodiments, the hGHpolypeptide further comprises the following substitutions: H18D, H₂₁N,R167N, K168A, D171S, K172R, E174S, I179T with a substitution at G120.(See, e.g., U.S. Pat. No. 5,849,535)

For the complete full-length naturally-occurring human GH amino acidsequence as well as the mature naturally-occurring GH amino acidsequence and naturally occurring mutant, see SEQ ID NO: 1, SEQ ID NO: 2and SEQ ID NO: 3, respectively, herein. In some embodiments, GHpolypeptides e.g., hGH polypeptides of the invention are substantiallyidentical to SEQ ID NO: 1, or SEQ ID NO: 2, or SEQ ID NO: 3 or any othersequence of a growth hormone polypeptide. For example, in someembodiments, GH polypeptides e.g., hGH polypeptides of the invention areat least about 60%, at least about 65%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95% or at least about 99% identical to SEQ ID NO: 1, orSEQ ID NO: 2, or SEQ ID NO: 3 or any other sequence of a growth hormonepolypeptide. In some embodiments, GH polypeptides e.g., hGH polypeptidesof the invention are at least about 60%, at least about 65%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95% or at least about 99% identicalto SEQ ID NO: 2. A number of naturally occurring mutants of hGH havebeen identified. These include hGH-V (Seeburg, DNA 1: 239 (1982); U.S.Pat. Nos. 4,446,235, 4,670,393, and 4,665,180, which are incorporated byreference herein) and a 20-kDa hGH containing a deletion of residues32-46 of hGH (SEQ ID NO: 3) (Kostyo et al., Biochem. Biophys. Acta 925:314 (1987); Lewis, U., et al., J. Biol. Chem., 253:2679-2687 (1978)).Placental growth hormone is described in Igout, A., et al., NucleicAcids Res. 17(10):3998 (1989)). In addition, numerous hGH variants,arising from post-transcriptional, post-translational, secretory,metabolic processing, and other physiological processes, have beenreported including proteolytically cleaved or 2 chain variants (Baumann,G., Endocrine Reviews 12: 424 (1991)). hGH dimers linked directly viaCys-Cys disulfide linkages are described in Lewis, U. J., et al., J.Biol. Chem. 252:3697-3702 (1977); Brostedt, P. and Roos, P., Prep.Biochem. 19:217-229 (1989)). Nucleic acid molecules encoding hGH mutantsand mutant hGH polypeptides are well known and include, but are notlimited to, those disclosed in U.S. Pat. Nos. 5,534,617; 5,580,723;5,688,666; 5,750,373; 5,834,250; 5,834,598; 5,849,535; 5,854,026;5,962,411; 5,955,346; 6,013,478; 6,022,711; 6,136,563; 6,143,523;6,428,954; 6,451,561; 6,780,613 and U.S. Patent Application Publication2003/0153003; which are incorporated by reference herein.

Commercial preparations of hGH are sold under the names: Humatrope™ (EliLilly & Co.), Nutropin™ (Genentech), Norditropin™ (Novo-Nordisk),Genotropin™ (Pfizer) and Saizen/Serostim™ (Serono).

The term “hGH polypeptide” also includes the pharmaceutically acceptablesalts and prodrugs, and prodrugs of the salts, polymorphs, hydrates,solvates, biologically-active fragments, biologically active variantsand stereoisomers of the naturally-occurring hGH as well as agonist,mimetic, and antagonist variants of the naturally-occurring hGH andpolypeptide fusions thereof. Fusions comprising additional amino acidsat the amino terminus, carboxyl terminus, or both, are encompassed bythe term “hGH polypeptide.” Exemplary fusions include, but are notlimited to, e.g., methionyl growth hormone in which a methionine islinked to the N-terminus of hGH resulting from the recombinantexpression of the mature form of hGH lacking the secretion signalpeptide or portion thereof, fusions for the purpose of purification(including, but not limited to, to poly-histidine or affinity epitopes),fusions with serum albumin binding peptides and fusions with serumproteins such as serum albumin. U.S. Pat. No. 5,750,373, which isincorporated by reference herein, describes a method for selecting novelproteins such as growth hormone and antibody fragment variants havingaltered binding properties for their respective receptor molecules. Themethod comprises fusing a gene encoding a protein of interest to thecarboxy terminal domain of the gene III coat protein of the filamentousphage M13.

Various references disclose modification of polypeptides by polymerconjugation or glycosylation. The term “hGH polypeptide” includespolypeptides conjugated to a polymer such as PEG and may be comprised ofone or more additional derivitizations of cysteine, lysine, or otherresidues. In addition, the hGH polypeptide may comprise a linker orpolymer, wherein the amino acid to which the linker or polymer isconjugated may be a non-natural amino acid according to the presentinvention, or may be conjugated to a naturally encoded amino acidutilizing techniques known in the art such as coupling to lysine orcysteine.

Polymer conjugation of hGH polypeptides has been reported. See, e.g.U.S. Pat. Nos. 5,849,535, 6,136,563 and 6,608,183, which areincorporated by reference herein. U.S. Pat. No. 4,904,584 disclosesPEGylated lysine depleted polypeptides, wherein at least one lysineresidue has been deleted or replaced with any other amino acid residue.WO 99/67291 discloses a process for conjugating a protein with PEG,wherein at least one amino acid residue on the protein is deleted andthe protein is contacted with PEG under conditions sufficient to achieveconjugation to the protein. WO 99/03887 discloses PEGylated variants ofpolypeptides belonging to the growth hormone superfamily, wherein acysteine residue has been substituted with a non-essential amino acidresidue located in a specified region of the polypeptide. WO 00/26354discloses a method of producing a glycosylated polypeptide variant withreduced allergenicity, which as compared to a corresponding parentpolypeptide comprises at least one additional glycosylation site. U.S.Pat. No. 5,218,092, which is incorporated by reference herein, disclosesmodification of granulocyte colony stimulating factor (G-CSF) and otherpolypeptides so as to introduce at least one additional carbohydratechain as compared to the native polypeptide.

The term “hGH polypeptide” also includes glycosylated hGH, as well asbut not limited to, polypeptides glycosylated at any amino acidposition, N-linked or O-linked glycosylated forms of the polypeptide.Variants containing single nucleotide changes are also considered asbiologically active variants of hGH polypeptide. In addition, splicevariants are also included. The term “hGH polypeptide” also includes GH,e.g., hGH polypeptide heterodimers, homodimers, heteromultimers, orhomomultimers of any one or more GH, e.g., hGH polypeptides or any otherpolypeptide, protein, carbohydrate, polymer, small molecule, linker,ligand, or other biologically active molecule of any type, linked bychemical means or expressed as a fusion protein, as well as polypeptideanalogues containing, for example, specific deletions or othermodifications yet maintain biological activity.

All references to amino acid positions in GH, e.g., hGH described hereinare based on the position in SEQ ID NO: 2, unless otherwise specified(i.e., when it is stated that the comparison is based on SEQ ID NO: 1,3, or other hGH sequence). Those of skill in the art will appreciatethat amino acid positions corresponding to positions in SEQ ID NO: 1, 2,3, or any other GH sequence can be readily identified in any other GH,e.g., hGH molecule such as GH, or hGH fusions, variants, fragments, etc.For example, sequence alignment programs such as BLAST can be used toalign and identify a particular position in a protein that correspondswith a position in SEQ ID NO: 1, 2, 3, or other GH sequence.Substitutions, deletions or additions of amino acids described herein inreference to SEQ ID NO: 1, 2, 3, or other GH sequence are intended toalso refer to substitutions, deletions or additions in correspondingpositions in GH, or hGH fusions, variants, fragments, etc. describedherein or known in the art and are expressly encompassed by the presentinvention.

The term “hGH polypeptide” or “hGH” encompasses hGH polypeptidescomprising one or more amino acid substitutions, additions or deletions.hGH polypeptides of the present invention may be comprised ofmodifications with one or more natural amino acids in conjunction withone or more non-natural amino acid modification. Exemplary substitutionsin a wide variety of amino acid positions in naturally-occurring hGHpolypeptides have been described, including but not limited tosubstitutions that modulate one or more of the biological activities ofthe hGH polypeptide, such as but not limited to, increase agonistactivity, increase solubility of the polypeptide, decrease proteasesusceptibility, convert the polypeptide into an antagonist, etc. and areencompassed by the term “hGH polypeptide.”

Human GH antagonists include, but are not limited to, those withsubstitutions at: 1, 2, 3, 4, 5, 8, 9, 11, 12, 15, 16, 19, 22, 103, 109,112, 113, 115, 116, 119, 120, 123, and 127 or an addition at position 1(i.e., at the N-terminus), or any combination thereof (SEQ ID NO:2, orthe corresponding amino acid in SEQ ID NO: 1, 3, or any other GHsequence). In some embodiments, hGH antagonists comprise at least onesubstitution in the regions 1-5 (N-terminus), 6-33 (A helix), 34-74(region between A helix and B helix, the A-B loop), 75-96 (B helix),97-105 (region between B helix and C helix, the B-C loop), 106-129 (Chelix), 130-153 (region between C helix and D helix, the C-D loop),154-183 (D helix), 184-191 (C-terminus) that cause GH to act as anantagonist. In other embodiments, the exemplary sites of incorporationof a non-naturally encoded amino acid include residues within the aminoterminal region of helix A and a portion of helix C. In anotherembodiment, substitution of G120 with a non-naturally encoded amino acidsuch as p-azido-L-phenyalanine or O-propargyl-L-tyrosine. In otherembodiments, the above-listed substitutions are combined with additionalsubstitutions that cause the hGH polypeptide to be an hGH antagonist.For instance, a non-naturally encoded amino acid is substituted at oneof the positions identified herein and a simultaneous substitution isintroduced at G120 (e.g., G120R, G120K, G120W, G120Y, G120F, or G120E).In some embodiments, the hGH antagonist comprises a non-naturallyencoded amino acid linked to a water soluble polymer that is present ina receptor binding region of the hGH molecule.

In some embodiments, the GH, e.g., hGH polypeptides further comprise anaddition, substitution or deletion that modulates biological activity ofthe GH or hGH polypeptide. For example, the additions, substitutions ordeletions may modulate one or more properties or activities of GH, e.g.,hGH. For example, the additions, substitutions or deletions may modulateaffinity for the GH, e.g., hGH polypeptide receptor, modulate (includingbut not limited to, increases or decreases) receptor dimerization,stabilize receptor dimers, circulating half-life, modulate therapeutichalf-life, modulate stability of the polypeptide, modulate cleavage byproteases, modulate dose, modulate release or bio-availability,facilitate purification, or improve or alter a particular route ofadministration. Similarly, GH, e.g., hGH polypeptides may compriseprotease cleavage sequences, reactive groups, antibody-binding domains(including but not limited to, FLAG or poly-His) or other affinity basedsequences (including but not limited to, FLAG, poly-His, GST, etc.) orlinked molecules (including but not limited to, biotin) that improvedetection (including but not limited to, GFP), purification or othertraits of the polypeptide.

The term “hGH polypeptide” also encompasses homodimers, heterodimers,homomultimers, and heteromultimers that are linked, including but notlimited to those linked directly via non-naturally encoded amino acidside chains, either to the same or different non-naturally encoded aminoacid side chains, to naturally-encoded amino acid side chains, orindirectly via a linker. Exemplary linkers including but are not limitedto, small organic compounds, water soluble polymers of a variety oflengths such as poly(ethylene glycol) or polydextran or polypeptides ofvarious lengths.

A “non-naturally encoded amino acid” refers to an amino acid that is notone of the common amino acids or pyrrolysine or selenocysteine. Otherterms that may be used synonymously with the term “non-naturally encodedamino acid” are “non-natural amino acid,” “unnatural amino acid,”“non-naturally-occurring amino acid,” and variously hyphenated andnon-hyphenated versions thereof. The term “non-naturally encoded aminoacid” also includes, but is not limited to, amino acids that occur bymodification (e.g. post-translational modifications) of a naturallyencoded amino acid (including but not limited to, the 20 common aminoacids or pyrrolysine and selenocysteine) but are not themselvesnaturally incorporated into a growing polypeptide chain by thetranslation complex. Examples of such non-naturally-occurring aminoacids include, but are not limited to, N-acetylglucosaminyl-L-serine,N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine.

An “amino terminus modification group” refers to any molecule that canbe attached to the amino terminus of a polypeptide. Similarly, a“carboxy terminus modification group” refers to any molecule that can beattached to the carboxy terminus of a polypeptide. Terminus modificationgroups include, but are not limited to, various water soluble polymers,peptides or proteins such as serum albumin, or other moieties thatincrease serum half-life of peptides.

The terms “functional group”, “active moiety”, “activating group”,“leaving group”, “reactive site”, “chemically reactive group” and“chemically reactive moiety” are used in the art and herein to refer todistinct, definable portions or units of a molecule. The terms aresomewhat synonymous in the chemical arts and are used herein to indicatethe portions of molecules that perform some function or activity and arereactive with other molecules.

The term “linkage” or “linker” is used herein to refer to groups orbonds that normally are formed as the result of a chemical reaction andtypically are covalent linkages. Hydrolytically stable linkages meansthat the linkages are substantially stable in water and do not reactwith water at useful pH values, including but not limited to, underphysiological conditions for an extended period of time, perhaps evenindefinitely. Hydrolytically unstable or degradable linkages mean thatthe linkages are degradable in water or in aqueous solutions, includingfor example, blood. Enzymatically unstable or degradable linkages meanthat the linkage can be degraded by one or more enzymes. As understoodin the art, PEG and related polymers may include degradable linkages inthe polymer backbone or in the linker group between the polymer backboneand one or more of the terminal functional groups of the polymermolecule. For example, ester linkages formed by the reaction of PEGcarboxylic acids or activated PEG carboxylic acids with alcohol groupson a biologically active agent generally hydrolyze under physiologicalconditions to release the agent. Other hydrolytically degradablelinkages include, but are not limited to, carbonate linkages; iminelinkages resulted from reaction of an amine and an aldehyde; phosphateester linkages formed by reacting an alcohol with a phosphate group;hydrazone linkages which are reaction product of a hydrazide and analdehyde; acetal linkages that are the reaction product of an aldehydeand an alcohol; orthoester linkages that are the reaction product of aformate and an alcohol; peptide linkages formed by an amine group,including but not limited to, at an end of a polymer such as PEG, and acarboxyl group of a peptide; and oligonucleotide linkages formed by aphosphoramidite group, including but not limited to, at the end of apolymer, and a 5′ hydroxyl group of an oligonucleotide.

The term “biologically active molecule”, “biologically active moiety” or“biologically active agent” when used herein means any substance whichcan affect any physical or biochemical properties of a biologicalsystem, pathway, molecule, or interaction relating to an organism,including but not limited to, viruses, bacteria, bacteriophage,transposon, prion, insects, fungi, plants, animals, and humans. Inparticular, as used herein, biologically active molecules include, butare not limited to, any substance intended for diagnosis, cure,mitigation, treatment, or prevention of disease in humans or otheranimals, or to otherwise enhance physical or mental well-being of humansor animals. Examples of biologically active molecules include, but arenot limited to, peptides, proteins, enzymes, small molecule drugs, harddrugs, soft drugs, carbohydrates, inorganic atoms or molecules, dyes,lipids, nucleosides, radionuclides, oligonucleotides, toxins, cells,viruses, liposomes, microparticles and micelles. Classes of biologicallyactive agents that are suitable for use with the invention include, butare not limited to, drugs, prodrugs, radionuclides, imaging agents,polymers, antibiotics, fungicides, anti-viral agents, anti-inflammatoryagents, anti-tumor agents, cardiovascular agents, anti-anxiety agents,hormones, growth factors, steroidal agents, microbially derived toxins,and the like.

A “bifunctional polymer” refers to a polymer comprising two discretefunctional groups that are capable of reacting specifically with othermoieties (including but not limited to, amino acid side groups) to formcovalent or non-covalent linkages. A bifunctional linker having onefunctional group reactive with a group on a particular biologicallyactive component, and another group reactive with a group on a secondbiological component, may be used to form a conjugate that includes thefirst biologically active component, the bifunctional linker and thesecond biologically active component. Many procedures and linkermolecules for attachment of various compounds to peptides are known.See, e.g., European Patent Application No. 188,256; U.S. Pat. Nos.4,671,958, 4,659,839, 4,414,148, 4,699,784; 4,680,338, and 4,569,789which are incorporated by reference herein. A “multi-functional polymer”refers to a polymer comprising two or more discrete functional groupsthat are capable of reacting specifically with other moieties (includingbut not limited to, amino acid side groups) to form covalent ornon-covalent linkages. A bi-functional polymer or multi-functionalpolymer may be any desired length or molecular weight, and may beselected to provide a particular desired spacing or conformation betweenone or more molecules linked to the GH e.g. hGH molecule.

Where substituent groups are specified by their conventional chemicalformulas, written from left to right, they equally encompass thechemically identical substituents that would result from writing thestructure from right to left, for example, the structure —CH₂O— isequivalent to the structure —OCH₂—.

The term “substituents” includes but is not limited to “non-interferingsubstituents”. “Non-interfering substituents” are those groups thatyield stable compounds. Suitable non-interfering substituents orradicals include, but are not limited to, halo, C₁-C₁₀ alkyl, C₂-C₁₀alkenyl, C₂-C₁₀ alkynyl, C₁-C₁₀ alkoxy, C₁-C₁₂ aralkyl, C₁-C₁₂ alkaryl,C₃-C₁₂ cycloalkyl, C₃-C₁₂ cycloalkenyl, phenyl, substituted phenyl,toluoyl, xylenyl, biphenyl, C₂-C₁₂ alkoxyalkyl, C₂-C₁₂ alkoxyaryl,C₇-C₁₂ aryloxyalkyl, C₇-C₁₂ oxyaryl, C₁-C₆ alkylsulfinyl, C₁-C₁₀alkylsulfonyl, —(CH₂)_(m)—O—(C₁-C₁₀ alkyl) wherein m is from 1 to 8,aryl, substituted aryl, substituted alkoxy, fluoroalkyl, heterocyclicradical, substituted heterocyclic radical, nitroalkyl, —NO₂, —CN,—NRC(O)—(C₁-C₁₀ alkyl), —C(O)—(C₁-C₁₀ alkyl), C₂-C₁₀ alkyl thioalkyl,—C(O)O—(C₁-C₁₀ alkyl), —OH, —SO₂, ═S, —COOH, —NR₂, carbonyl,—C(O)—(C₁-C₁₀ alkyl)-CF₃, —C(O)—CF3, —C(O)NR2, —(C₁-C₁₀ aryl)-S—(C₆-C₁₀aryl), —C(O)—(C₁-C₁₀ aryl), —(CH₂)_(m)—O—(—(CH₂)_(m)—O—(C₁-C₁₀ alkyl)wherein each m is from 1 to 8, —C(O)NR₂, —C(S)NR₂, —SO₂NR₂, —NRC(O)NR₂,—NRC(S)NR₂, salts thereof, and the like. Each R as used herein is H,alkyl or substituted alkyl, aryl or substituted aryl, aralkyl, oralkaryl.

The term “halogen” includes fluorine, chlorine, iodine, and bromine.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include di- and multivalentradicals, having the number of carbon atoms designated (i.e. C₁-C₁₀means one to ten carbons). Examples of saturated hydrocarbon radicalsinclude, but are not limited to, groups such as methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,(cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Anunsaturated alkyl group is one having one or more double bonds or triplebonds. Examples of unsaturated alkyl groups include, but are not limitedto, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,3-butynyl, and the higher homologs and isomers. The term “alkyl,” unlessotherwise noted, is also meant to include those derivatives of alkyldefined in more detail below, such as “heteroalkyl.” Alkyl groups whichare limited to hydrocarbon groups are termed “homoalkyl”.

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from an alkane, as exemplified, but notlimited, by the structures —CH₂CH₂— and —CH₂CH₂CH₂CH₂—, and furtherincludes those groups described below as “heteroalkylene.” Typically, analkyl (or alkylene) group will have from 1 to 24 carbon atoms, withthose groups having 10 or fewer carbon atoms being a particularembodiment of the methods and compositions described herein. A “loweralkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group,generally having eight or fewer carbon atoms.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of thestated number of carbon atoms and at least one heteroatom selected fromthe group consisting of O, N, Si and S, and wherein the nitrogen andsulfur atoms may optionally be oxidized and the nitrogen heteroatom mayoptionally be quaternized. The heteroatom(s) O, N and S and Si may beplaced at any interior position of the heteroalkyl group or at theposition at which the alkyl group is attached to the remainder of themolecule. Examples include, but are not limited to, —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂,—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃,and —CH═CH—N(CH₃)—CH₃. Up to two heteroatoms may be consecutive, suchas, for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃. Similarly, the term“heteroalkylene” by itself or as part of another substituent means adivalent radical derived from heteroalkyl, as exemplified, but notlimited by, —CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. Forheteroalkylene groups, the same or different heteroatoms can also occupyeither or both of the chain termini (including but not limited to,alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino,aminooxyalkylene, and the like). Still further, for alkylene andheteroalkylene linking groups, no orientation of the linking group isimplied by the direction in which the formula of the linking group iswritten. For example, the formula —C(O)₂R′— represents both —C(O)₂R′—and —R′C(O)₂—.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Thus, a cycloalkylor heterocycloalkyl may include saturated, partially and fullyunsaturated ring linkages. Additionally, for heterocycloalkyl, aheteroatom can occupy the position at which the heterocycle is attachedto the remainder of the molecule. Examples of cycloalkyl include, butare not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl,3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkylinclude, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl),1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl,3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl,tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl,2-piperazinyl, and the like. Additionally, the term encompasses bicyclicand tricyclic ring structures. Similarly, the term “heterocycloalkylene”by itself or as part of another substituent means a divalent radicalderived from heterocycloalkyl, and the term “cycloalkylene” by itself oras part of another substituent means a divalent radical derived fromcycloalkyl.

As used herein; the term “water soluble polymer” refers to any polymerthat is soluble in aqueous solvents. Linkage of water soluble polymersto GH, e.g., hGH polypeptides can result in changes including, but notlimited to, increased or modulated serum half-life, or increased ormodulated therapeutic half-life relative to the unmodified form,modulated immunogenicity, modulated physical association characteristicssuch as aggregation and multimer formation, altered receptor binding,and altered receptor dimerization or multimerization. The water solublepolymer may or may not have its own biological activity, and may beutilized as a linker for attaching GH, e.g., hGH to other substances,including but not limited to one or more GH, e.g., hGH polypeptides, orone or more biologically active molecules. Suitable polymers include,but are not limited to, polyethylene glycol, polyethylene glycolpropionaldehyde, mono C1-C10 alkoxy or aryloxy derivatives thereof(described in U.S. Pat. No. 5,252,714 which is incorporated by referenceherein), monomethoxy-polyethylene glycol, polyvinyl pyrrolidone,polyvinyl alcohol, polyamino acids, divinylether maleic anhydride,N-(2-Hydroxypropyl)-methacrylamide, dextran, dextran derivativesincluding dextran sulfate, polypropylene glycol, polypropyleneoxide/ethylene oxide copolymer, polyoxyethylated polyol, heparin,heparin fragments, polysaccharides, oligosaccharides, glycans, celluloseand cellulose derivatives, including but not limited to methylcelluloseand carboxymethyl cellulose, starch and starch derivatives,polypeptides, polyalkylene glycol and derivatives thereof, copolymers ofpolyalkylene glycols and derivatives thereof, polyvinyl ethyl ethers,and alpha-beta-poly[(2-hydroxyethyl)-DL-aspartamide, and the like, ormixtures thereof. Examples of such water soluble polymers include, butare not limited to, polyethylene glycol and serum albumin.

As used herein, the term “polyalkylene glycol” or “poly(alkene glycol)”refers to polyethylene glycol (poly(ethylene glycol)), polypropyleneglycol, polybutylene glycol, and derivatives thereof. The term“polyalkylene glycol” and/or “polyethylene glycol” encompasses bothlinear and branched polymers and average molecular weights of between0.1 kDa and 100 kDa. Other exemplary embodiments are listed, forexample, in commercial supplier catalogs, such as ShearwaterCorporation's catalog “Polyethylene Glycol and Derivatives forBiomedical Applications” (2001).

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent which can be a single ring or multiplerings (including but not limited to, from 1 to 3 rings) which are fusedtogether or linked covalently. The term “heteroaryl” refers to arylgroups (or rings) that contain from one to four heteroatoms selectedfrom N, O, and S, wherein the nitrogen and sulfur atoms are optionallyoxidized, and the nitrogen atom(s) are optionally quaternized. Aheteroaryl group can be attached to the remainder of the moleculethrough a heteroatom. Non-limiting examples of aryl and heteroarylgroups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl,2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl,pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl,3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl,5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl,3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl,purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl,2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituentsfor each of the above noted aryl and heteroaryl ring systems areselected from the group of acceptable substituents described below.

For brevity, the term “aryl” when used in combination with other terms(including but not limited to, aryloxy, arylthioxy, arylalkyl) includesboth aryl and heteroaryl rings as defined above. Thus, the term“arylalkyl” is meant to include those radicals in which an aryl group isattached to an alkyl group (including but not limited to, benzyl,phenethyl, pyridylmethyl and the like) including those alkyl groups inwhich a carbon atom (including but not limited to, a methylene group)has been replaced by, for example, an oxygen atom (including but notlimited to, phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl,and the like).

Each of the above terms (including but not limited to, “alkyl,”“heteroalkyl,” “aryl” and “heteroaryl”) are meant to include bothsubstituted and unsubstituted forms of the indicated radical. Exemplarysubstituents for each type of radical are provided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂ in a number ranging from zero to (2m′+1), where m′ is the totalnumber of carbon atoms in such a radical. R′, R″, R′″ and R″″ eachindependently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, including but notlimited to, aryl substituted with 1-3 halogens, substituted orunsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups.When a compound of the invention includes more than one R group, forexample, each of the R groups is independently selected as are each R′,R″, R′″ and R″″ groups when more than one of these groups is present.When R′ and R″ are attached to the same nitrogen atom, they can becombined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.For example, —NR′R″ is meant to include, but not be limited to,1-pyrrolidinyl and 4-morpholinyl. From the above discussion ofsubstituents, one of skill in the art will understand that the term“alkyl” is meant to include groups including carbon atoms bound togroups other than hydrogen groups, such as haloalkyl (including but notlimited to, —CF₃ and —CH₂CF₃) and acyl (including but not limited to,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are varied and areselected from, but are not limited to: halogen, —OR′, ═O, ═NR′, ═N—OR′,—NR′R″, —SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″,—OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′,—NR—C(NR′R″R″″)═NR″″, —NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″,—NRSO₂R′, —CN and —NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, andfluoro(C₁-C₄)alkyl, in a number ranging from zero to the total number ofopen valences on the aromatic ring system; and where R′, R″, R′″ and R″″are independently selected from hydrogen, alkyl, heteroalkyl, aryl andheteroaryl. When a compound of the invention includes more than one Rgroup, for example, each of the R groups is independently selected asare each R′, R″, R′″ and R″″ groups when more than one of these groupsis present.

As used herein, the term “modulated serum half-life” means the positiveor negative change in circulating half-life of a modified hGH relativeto its non-modified form. Serum half-life is measured by taking bloodsamples at various time points after administration of hGH, anddetermining the concentration of that molecule in each sample.Correlation of the serum concentration with time allows calculation ofthe serum half-life. Increased serum half-life desirably has at leastabout two-fold, but a smaller increase may be useful, for example whereit enables a satisfactory dosing regimen or avoids a toxic effect. Insome embodiments, the increase is at least about three-fold, at leastabout five-fold, or at least about ten-fold.

The term “modulated therapeutic half-life” as used herein means thepositive or negative change in the half-life of the therapeuticallyeffective amount of a modified hGH, relative to its non-modified form.Therapeutic half-life is measured by measuring pharmacokinetic and/orpharmacodynamic properties of the molecule at various time points afteradministration. Increased therapeutic half-life desirably enables aparticular beneficial dosing regimen, a particular beneficial totaldose, or avoids an undesired effect. In some embodiments, the increasedtherapeutic half-life results from increased potency, increased ordecreased binding of the modified molecule to its target, increased ordecreased breakdown of the molecule by enzymes such as proteases, or anincrease or decrease in another parameter or mechanism of action of thenon-modified molecule.

The term “isolated,” when applied to a nucleic acid or protein, denotesthat the nucleic acid or protein is free of at least some of thecellular components with which it is associated in the natural state, orthat the nucleic acid or protein has been concentrated to a levelgreater than the concentration of its in vivo or in vitro production. Itcan be in a homogeneous state. Isolated substances can be in either adry or semi-dry state, or in solution, including but not limited to, anaqueous solution. It can be a component of a pharmaceutical compositionthat comprises additional pharmaceutically acceptable carriers and/orexcipients. Purity and homogeneity are typically determined usinganalytical chemistry techniques such as polyacrylamide gelelectrophoresis or high performance liquid chromatography. A proteinwhich is the predominant species present in a preparation issubstantially purified. In particular, an isolated gene is separatedfrom open reading frames which flank the gene and encode a protein otherthan the gene of interest. The term “purified” denotes that a nucleicacid or protein gives rise to substantially one band in anelectrophoretic gel. Particularly, it may mean that the nucleic acid orprotein is at least 85% pure, at least 90% pure, at least 95% pure, atleast 99% or greater pure.

The term “nucleic acid” refers to deoxyribonucleotides,deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymersthereof in either single- or double-stranded form. Unless specificallylimited, the term encompasses nucleic acids containing known analoguesof natural nucleotides which have similar binding properties as thereference nucleic acid and are metabolized in a manner similar tonaturally occurring nucleotides. Unless specifically limited otherwise,the term also refers to oligonucleotide analogs including PNA(peptidonucleic acid), analogs of DNA used in antisense technology(phosphorothioates, phosphoroamidates, and the like). Unless otherwiseindicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof (including but notlimited to, degenerate codon substitutions) and complementary sequencesas well as the sequence explicitly indicated. Specifically, degeneratecodon substitutions may be achieved by generating sequences in which thethird position of one or more selected (or all) codons is substitutedwith mixed-base and/or deoxyinosine residues (Batzer et al., NucleicAcid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues.That is, a description directed to a polypeptide applies equally to adescription of a peptide and a description of a protein, and vice versa.The terms apply to naturally occurring amino acid polymers as well asamino acid polymers in which one or more amino acid residues is anon-naturally encoded amino acid. As used herein, the terms encompassamino acid chains of any length, including full length proteins, whereinthe amino acid residues are linked by covalent peptide bonds.

The term “amino acid” refers to naturally occurring and non-naturallyoccurring amino acids, as well as amino acid analogs and amino acidmimetics that function in a manner similar to the naturally occurringamino acids. Naturally encoded amino acids are the 20 common amino acids(alanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, and valine) and pyrrolysine and selenocysteine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an α carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, such as,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (such as, norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, “conservatively modified variants” refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of ordinary skill inthe art will recognize that each codon in a nucleic acid (except AUG,which is ordinarily the only codon for methionine, and TGG, which isordinarily the only codon for tryptophan) can be modified to yield afunctionally identical molecule. Accordingly, each silent variation of anucleic acid which encodes a polypeptide is implicit in each describedsequence.

As to amino acid sequences, one of ordinary skill in the art willrecognize that individual substitutions, deletions or additions to anucleic acid, peptide, polypeptide, or protein sequence which alters,adds or deletes a single amino acid or a small percentage of amino acidsin the encoded sequence is a “conservatively modified variant” where thealteration results in the deletion of an amino acid, addition of anamino acid, or substitution of an amino acid with a chemically similaramino acid. Conservative substitution tables providing functionallysimilar amino acids are known to those of ordinary skill in the art.Such conservatively modified variants are in addition to and do notexclude polymorphic variants, interspecies homologs, and alleles of theinvention.

Conservative substitution tables providing functionally similar aminoacids are known to those of ordinary skill in the art. The followingeight groups each contain amino acids that are conservativesubstitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

7) Serine (S), Threonine (T); and

8) Cysteine (C), Methionine (M)

(see, e.g., Creighton, Proteins: Structures and Molecular Properties (WH Freeman & Co.; 2nd edition (December 1993)

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same. Sequences are“substantially identical” if they have a percentage of amino acidresidues or nucleotides that are the same (i.e., at least about 60%identity, at least about 65%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, or at least about 99% identity over a specified region), whencompared and aligned for maximum correspondence over a comparisonwindow, or designated region as measured using one of the followingsequence comparison algorithms (or other algorithms available to personsof ordinary skill in the art) or by manual alignment and visualinspection. This definition also refers to the complement of a testsequence. The identity can exist over a region that is at least about 50amino acids or nucleotides in length, or over a region that is 75-100amino acids or nucleotides in length, or, where not specified, acrossthe entire sequence of a polynucleotide or polypeptide.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are known to those of ordinary skill in the art. Optimalalignment of sequences for comparison can be conducted, including butnot limited to, by the local homology algorithm of Smith and Waterman(1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search forsimilarity method of Pearson and Lipman (1988) Proc. Nat'l. Acad. Sci.USA 85:2444, by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manualalignment and visual inspection (see, e.g., Ausubel et al., CurrentProtocols in Molecular Biology (1995 supplement)).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1997) Nuc. AcidsRes. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410,respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Informationavailable at the World Wide Web at ncbi.nlm.nih.gov. The BLAST algorithmparameters W, T, and X determine the sensitivity and speed of thealignment. The BLASTN program (for nucleotide sequences) uses asdefaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=−4 anda comparison of both strands. For amino acid sequences, the BLASTPprogram uses as defaults a wordlength of 3, and expectation (E) of 10,and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc.Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation (E) of10, M=5, N=−4, and a comparison of both strands. The BLAST algorithm istypically performed with the “low complexity” filter turned off.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid may be less than about 0.2, or less thanabout 0.01, or less than about 0.001.

The phrase “selectively (or specifically) hybridizes to” refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence under stringent hybridization conditions when thatsequence is present in a complex mixture (including but not limited to,total cellular or library DNA or RNA).

The phrase “stringent hybridization conditions” refers to hybridizationof sequences of DNA, RNA, PNA, or other nucleic acid mimics, orcombinations thereof under conditions of low ionic strength and hightemperature as is known in the art. Typically, under stringentconditions a probe will hybridize to its target subsequence in a complexmixture of nucleic acid (including but not limited to, total cellular orlibrary DNA or RNA) but does not hybridize to other sequences in thecomplex mixture. Stringent conditions are sequence-dependent and will bedifferent in different circumstances. Longer sequences hybridizespecifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen, LaboratoryTechniques in Biochemistry and Molecular Biology—Hybridization withNucleic Probes, “Overview of principles of hybridization and thestrategy of nucleic acid assays” (1993). Generally, stringent conditionsare selected to be about 5-10° C. lower than the thermal melting point(T_(m)) for the specific sequence at a defined ionic strength pH. TheT_(m) is the temperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at T_(m), 50% of the probes are occupied atequilibrium). Stringent conditions may be those in which the saltconcentration is less than about 1.0 M sodium ion, typically about 0.01to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 andthe temperature is at least about 30° C. for short probes (including butnot limited to, 10 to 50 nucleotides) and at least about 60° C. for longprobes (including but not limited to, greater than 50 nucleotides).Stringent conditions may also be achieved with the addition ofdestabilizing agents such as formamide. For selective or specifichybridization, a positive signal may be at least two times background,optionally 10 times background hybridization. Exemplary stringenthybridization conditions can be as following: 50% formamide, 5×SSC, and1% SDS, incubating at 42° C., or 5×SSC, 1% SDS, incubating at 65° C.,with wash in 0.2×SSC, and 0.1% SDS at 65° C. Such washes can beperformed for 5, 15, 30, 60, 120, or more minutes.

As used herein, the term “eukaryote” refers to organisms belonging tothe phylogenetic domain Eucarya such as animals (including but notlimited to, mammals, insects, reptiles, birds, etc.), ciliates, plants(including but not limited to, monocots, dicots, algae, etc.), fungi,yeasts, flagellates, microsporidia, protists, etc.

As used herein, the term “non-eukaryote” refers to non-eukaryoticorganisms. For example, a non-eukaryotic organism can belong to theEubacteria (including but not limited to, Escherichia coli, Thermusthermophilus, Bacillus stearothermophilus, Pseudomonas fluorescens,Pseudomonas aeruginosa, Pseudomonas putida, etc.) phylogenetic domain,or the Archaea (including but not limited to, Methanococcus jannaschii,Methanobacterium thermoautotrophicum, Halobacterium such as Haloferaxvolcanii and Halobacterium species NRC-1, Archaeoglobus fulgidus,Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum pernix, etc.)phylogenetic domain.

The term “subject” as used herein, refers to an animal, in someembodiments a mammal, and in other embodiments a human, who is theobject of treatment, observation or experiment.

The term “effective amount” as used herein refers to that amount of themodified non-natural amino acid polypeptide being administered whichwill relieve to some extent one or more of the symptoms of the disease,condition or disorder being treated. Compositions containing themodified non-natural amino acid polypeptide described herein can beadministered for prophylactic, enhancing, and/or therapeutic treatments.

The terms “enhance” or “enhancing” means to increase or prolong eitherin potency or duration a desired effect. Thus, in regard to enhancingthe effect of therapeutic agents, the term “enhancing” refers to theability to increase or prolong, either in potency or duration, theeffect of other therapeutic agents on a system. An “enhancing-effectiveamount,” as used herein, refers to an amount adequate to enhance theeffect of another therapeutic agent in a desired system. When used in apatient, amounts effective for this use will depend on the severity andcourse of the disease, disorder or condition, previous therapy, thepatient's health status and response to the drugs, and the judgment ofthe treating physician.

The term “modified,” as used herein refers to any changes made to agiven polypeptide, such as changes to the length of the polypeptide, theamino acid sequence, chemical structure, co-translational modification,or post-translational modification of a polypeptide. The form“(modified)” term means that the polypeptides being discussed areoptionally modified, that is, the polypeptides under discussion can bemodified or unmodified.

The term “post-translationally modified” refers to any modification of anatural or non-natural amino acid that occurs to such an amino acidafter it has been incorporated into a polypeptide chain. The termencompasses, by way of example only, co-translational in vivomodifications, co-translational in vitro modifications (such as in acell-free translation system), post-translational in vivo modifications,and post-translational in vitro modifications.

In prophylactic applications, compositions containing the modifiednon-natural amino acid polypeptide are administered to a patientsusceptible to or otherwise at risk of a particular disease, disorder orcondition. Such an amount is defined to be a “prophylactically effectiveamount.” In this use, the precise amounts also depend on the patient'sstate of health, weight, and the like. It is considered well within theskill of the art for one to determine such prophylactically effectiveamounts by routine experimentation (e.g., a dose escalation clinicaltrial).

The term “protected” refers to the presence of a “protecting group” ormoiety that prevents reaction of the chemically reactive functionalgroup under certain reaction conditions. The protecting group will varydepending on the type of chemically reactive group being protected. Forexample, if the chemically reactive group is an amine or a hydrazide,the protecting group can be selected from the group oftert-butyloxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc). Ifthe chemically reactive group is a thiol, the protecting group can beorthopyridyldisulfide. If the chemically reactive group is a carboxylicacid, such as butanoic or propionic acid, or a hydroxyl group, theprotecting group can be benzyl or an alkyl group such as methyl, ethyl,or tert-butyl. Other protecting groups known in the art may also be usedin or with the methods and compositions described herein, includingphotolabile groups such as Nvoc and MeNvoc. Other protecting groupsknown in the art may also be used in or with the methods andcompositions described herein.

By way of example only, blocking/protecting groups may be selected from:

Other protecting groups are described in Greene and Wuts, ProtectiveGroups in Organic Synthesis, 3rd Ed., John Wiley & Sons, New York, N.Y.,1999, which is incorporated herein by reference in its entirety.

In therapeutic applications, compositions containing the modifiednon-natural amino acid polypeptide are administered to a patient alreadysuffering from a disease, condition or disorder, in an amount sufficientto cure or at least partially arrest the symptoms of the disease,disorder or condition. Such an amount is defined to be a“therapeutically effective amount,” and will depend on the severity andcourse of the disease, disorder or condition, previous therapy, thepatient's health status and response to the drugs, and the judgment ofthe treating physician. It is considered well within the skill of theart for one to determine such therapeutically effective amounts byroutine experimentation (e.g., a dose escalation clinical trial).

The term “treating” is used to refer to either prophylactic and/ortherapeutic treatments.

Non-naturally encoded amino acid polypeptides presented herein mayinclude isotopically-labelled compounds with one or more atoms replacedby an atom having an atomic mass or mass number different from theatomic mass or mass number usually found in nature. Examples of isotopesthat can be incorporated into the present compounds include isotopes ofhydrogen, carbon, nitrogen, oxygen, fluorine and chlorine, such as ²H,³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³⁵S, ¹⁸F, ³⁶Cl, respectively. Certainisotopically-labelled compounds described herein, for example those intowhich radioactive isotopes such as ³H and ¹⁴C are incorporated, may beuseful in drug and/or substrate tissue distribution assays. Further,substitution with isotopes such as deuterium, i.e., 2H, can affordcertain therapeutic advantages resulting from greater metabolicstability, for example increased in vivo half-life or reduced dosagerequirements.

All isomers including but not limited to diastereomers, enantiomers, andmixtures thereof are considered as part of the compositions describedherein. In additional or further embodiments, the non-naturally encodedamino acid polypeptides are metabolized upon administration to anorganism in need to produce a metabolite that is then used to produce adesired effect, including a desired therapeutic effect. In further oradditional embodiments are active metabolites of non-naturally encodedamino acid polypeptides.

In some situations, non-naturally encoded amino acid polypeptides mayexist as tautomers. In addition, the non-naturally encoded amino acidpolypeptides described herein can exist in unsolvated as well assolvated forms with pharmaceutically acceptable solvents such as water,ethanol, and the like. The solvated forms are also considered to bedisclosed herein. Those of ordinary skill in the art will recognize thatsome of the compounds herein can exist in several tautomeric forms. Allsuch tautomeric forms are considered as part of the compositionsdescribed herein.

Unless otherwise indicated, conventional methods of mass spectroscopy,NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniquesand pharmacology, within the skill of the art are employed.

DETAILED DESCRIPTION

I. Introduction

GH, e.g., hGH molecules comprising at least one unnatural amino acid areprovided in the invention. In certain embodiments of the invention, theGH, e.g., hGH polypeptide with at least one unnatural amino acidincludes at least one post-translational modification. In oneembodiment, the at least one post-translational modification comprisesattachment of a molecule including but not limited to, a label, a dye, apolymer, a water-soluble polymer, a derivative of polyethylene glycol, aphotocrosslinker, a radionuclide, a cytotoxic compound, a drug, anaffinity label, a photoaffinity label, a reactive compound, a resin, asecond protein or polypeptide or polypeptide analog, an antibody orantibody fragment, a metal chelator, a cofactor, a fatty acid, acarbohydrate, a polynucleotide, a DNA, a RNA, an antisensepolynucleotide, a saccharide, water-soluble dendrimer, a cyclodextrin,an inhibitory ribonucleic acid, a biomaterial, a nanoparticle, a spinlabel, a fluorophore, a metal-containing moiety, a radioactive moiety, anovel functional group, a group that covalently or noncovalentlyinteracts with other molecules, a photocaged moiety, an actinicradiation excitable moiety, a photoisomerizable moiety, biotin, aderivative of biotin, a biotin analogue, a moiety incorporating a heavyatom, a chemically cleavable group, a photocleavable group, an elongatedside chain, a carbon-linked sugar, a redox-active agent, an aminothioacid, a toxic moiety, an isotopically labeled moiety, a biophysicalprobe, a phosphorescent group, a chemiluminescent group, an electrondense group, a magnetic group, an intercalating group, a chromophore, anenergy transfer agent, a biologically active agent, a detectable label,a small molecule, a quantum dot, a nanotransmitter, a radionucleotide, aradiotransmitter, a neutron-capture agent, or any combination of theabove or any other desirable compound or substance, comprising a secondreactive group to at least one unnatural amino acid comprising a firstreactive group utilizing chemistry methodology that is known to one ofordinary skill in the art to be suitable for the particular reactivegroups. For example, the first reactive group is an alkynyl moiety(including but not limited to, in the unnatural amino acidp-propargyloxyphenylalanine, where the propargyl group is also sometimesreferred to as an acetylene moiety) and the second reactive group is anazido moiety, and [3+2]cycloaddition chemistry methodologies areutilized. In another example, the first reactive group is the azidomoiety (including but not limited to, in the unnatural amino acidp-azido-L-phenylalanine) and the second reactive group is the alkynylmoiety. In certain embodiments of the modified GH, e.g., hGH polypeptideof the present invention, at least one unnatural amino acid (includingbut not limited to, unnatural amino acid containing a keto functionalgroup) comprising at least one post-translational modification, is usedwhere the at least one post-translational modification comprises asaccharide moiety. In certain embodiments, the post-translationalmodification is made in vivo in a eukaryotic cell or in a non-eukaryoticcell.

In certain embodiments, the protein includes at least onepost-translational modification that is made in vivo by one host cell,where the post-translational modification is not normally made byanother host cell type. In certain embodiments, the protein includes atleast one post-translational modification that is made in vivo by aeukaryotic cell, where the post-translational modification is notnormally made by a non-eukaryotic cell. Examples of post-translationalmodifications include, but are not limited to, glycosylation,acetylation, acylation, lipid-modification, palmitoylation, palmitateaddition, phosphorylation, glycolipid-linkage modification, and thelike. In one embodiment, the post-translational modification comprisesattachment of an oligosaccharide to an asparagine by a GlcNAc-asparaginelinkage (including but not limited to, where the oligosaccharidecomprises (GlcNAc-Man)₂-Man-GlcNAc-GlcNAc, and the like). In anotherembodiment, the post-translational modification comprises attachment ofan oligosaccharide (including but not limited to, Gal-GalNAc,Gal-GlcNAc, etc.) to a serine or threonine by a GalNAc-serine, aGalNAc-threonine, a GlcNAc-serine, or a GlcNAc-threonine linkage. Incertain embodiments, a protein or polypeptide of the invention cancomprise a secretion or localization sequence, an epitope tag, a FLAGtag, a polyhistidine tag, a GST fusion, and/or the like. Examples ofsecretion signal sequences include, but are not limited to, aprokaryotic secretion signal sequence, a eukaryotic secretion signalsequence, a eukaryotic secretion signal sequence 5′-optimized forbacterial expression, a novel secretion signal sequence, pectate lyasesecretion signal sequence, Omp A secretion signal sequence, and a phagesecretion signal sequence. Examples of secretion signal sequences,include, but are not limited to, STII (prokaryotic), Fd GIII and M13(phage), Bgl2 (yeast), and the signal sequence bla derived from atransposon.

The protein or polypeptide of interest can contain at least one, atleast two, at least three, at least four, at least five, at least six,at least seven, at least eight, at least nine, or ten or more unnaturalamino acids. The unnatural amino acids can be the same or different, forexample, there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more differentsites in the protein that comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moredifferent unnatural amino acids. In certain embodiments, at least one,but fewer than all, of a particular amino acid present in a naturallyoccurring version of the protein is substituted with an unnatural aminoacid.

The present invention provides methods and compositions based on membersof the GH supergene family, in particular hGH, comprising at least onenon-naturally encoded amino acid. Introduction of at least onenon-naturally encoded amino acid into a GH supergene family member canallow for the application of conjugation chemistries that involvespecific chemical reactions, including, but not limited to, with one ormore non-naturally encoded amino acids while not reacting with thecommonly occurring 20 amino acids. In some embodiments, the GH supergenefamily member comprising the non-naturally encoded amino acid is linkedto a water soluble polymer, such as polyethylene glycol (PEG), via theside chain of the non-naturally encoded amino acid. This inventionprovides a highly efficient method for the selective modification ofproteins with PEG derivatives, which involves the selectiveincorporation of non-genetically encoded amino acids, including but notlimited to, those amino acids containing functional groups orsubstituents not found in the 20 naturally incorporated amino acids,including but not limited to a ketone, an azide or acetylene moiety,into proteins in response to a selector codon and the subsequentmodification of those amino acids with a suitably reactive PEGderivative. Once incorporated, the amino acid side chains can then bemodified by utilizing chemistry methodologies known to those of ordinaryskill in the art to be suitable for the particular functional groups orsubstituents present in the non-naturally encoded amino acid. Knownchemistry methodologies of a wide variety are suitable for use in thepresent invention to incorporate a water soluble polymer into theprotein. Such methodologies include but are not limited to a Huisgen[3+2]cycloaddition reaction (see, e.g., Padwa, A. in ComprehensiveOrganic Synthesis, Vol. 4, (1991) Ed. Trost, B. M., Pergamon, Oxford, p.1069-1109; and, Huisgen, R. in 1,3-Dipolar Cycloaddition Chemistry,(1984) Ed. Padwa, A., Wiley, New York, p. 1-176) with, including but notlimited to, acetylene or azide derivatives, respectively.

Because the Huisgen [3+2]cycloaddition method involves a cycloadditionrather than a nucleophilic substitution reaction, proteins can bemodified with extremely high selectivity. The reaction can be carriedout at room temperature in aqueous conditions with excellentregioselectivity (1,4>1,5) by the addition of catalytic amounts of Cu(I)salts to the reaction mixture. See, e.g., Tornoe, et al., (2002) J. Org.Chem. 67:3057-3064; and, Rostovtsev, et al., (2002) Angew. Chem. Int.Ed. 41:2596-2599; and WO 03/101972. A molecule that can be added to aprotein of the invention through a [3+2]cycloaddition includes virtuallyany molecule with a suitable functional group or substituent includingbut not limited to an azido or acetylene derivative. These molecules canbe added to an unnatural amino acid with an acetylene group, includingbut not limited to, p-propargyloxyphenylalanine, or azido group,including but not limited to p-azido-phenylalanine, respectively.

The five-membered ring that results from the Huisgen [3+2]cycloadditionis not generally reversible in reducing environments and is stableagainst hydrolysis for extended periods in aqueous environments.Consequently, the physical and chemical characteristics of a widevariety of substances can be modified under demanding aqueous conditionswith the active PEG derivatives of the present invention. Even moreimportant, because the azide and acetylene moieties are specific for oneanother (and do not, for example, react with any of the 20 common,genetically-encoded amino acids), proteins can be modified in one ormore specific sites with extremely high selectivity.

The invention also provides water soluble and hydrolytically stablederivatives of PEG derivatives and related hydrophilic polymers havingone or more acetylene or azide moieties. The PEG polymer derivativesthat contain acetylene moieties are highly selective for coupling withazide moieties that have been introduced selectively into proteins inresponse to a selector codon. Similarly, PEG polymer derivatives thatcontain azide moieties are highly selective for coupling with acetylenemoieties that have been introduced selectively into proteins in responseto a selector codon.

More specifically, the azide moieties comprise, but are not limited to,alkyl azides, aryl azides and derivatives of these azides. Thederivatives of the alkyl and aryl azides can include other substituentsso long as the acetylene-specific reactivity is maintained. Theacetylene moieties comprise alkyl and aryl acetylenes and derivatives ofeach. The derivatives of the alkyl and aryl acetylenes can include othersubstituents so long as the azide-specific reactivity is maintained.

The present invention provides conjugates of substances having a widevariety of functional groups, substituents or moieties, with othersubstances including but not limited to a label; a dye; a polymer; awater-soluble polymer; a derivative of polyethylene glycol; aphotocrosslinker; a radionuclide; a cytotoxic compound; a drug; anaffinity label; a photoaffinity label; a reactive compound; a resin; asecond protein or polypeptide or polypeptide analog; an antibody orantibody fragment; a metal chelator; a cofactor; a fatty acid; acarbohydrate; a polynucleotide; a DNA; a RNA; an antisensepolynucleotide; a saccharide; a water-soluble dendrimer; a cyclodextrin;an inhibitory ribonucleic acid; a biomaterial; a nanoparticle; a spinlabel; a fluorophore, a metal-containing moiety; a radioactive moiety; anovel functional group; a group that covalently or noncovalentlyinteracts with other molecules; a photocaged moiety; an actinicradiation excitable moiety; a photoisomerizable moiety; biotin; aderivative of biotin; a biotin analogue; a moiety incorporating a heavyatom; a chemically cleavable group; a photocleavable group; an elongatedside chain; a carbon-linked sugar; a redox-active agent; an aminothioacid; a toxic moiety; an isotopically labeled moiety; a biophysicalprobe; a phosphorescent group; a chemiluminescent group; an electrondense group; a magnetic group; an intercalating group; a chromophore; anenergy transfer agent; a biologically active agent; a detectable label;a small molecule; a quantum dot; a nanotransmitter; a radionucleotide; aradiotransmitter; a neutron-capture agent; or any combination of theabove, or any other desirable compound or substance. The presentinvention also includes conjugates of substances having azide oracetylene moieties with PEG polymer derivatives having the correspondingacetylene or azide moieties. For example, a PEG polymer containing anazide moiety can be coupled to a biologically active molecule at aposition in the protein that contains a non-genetically encoded aminoacid bearing an acetylene functionality. The linkage by which the PEGand the biologically active molecule are coupled includes but is notlimited to the Huisgen [3+2]cycloaddition product.

It is well established in the art that PEG can be used to modify thesurfaces of biomaterials (see, e.g., U.S. Pat. No. 6,610,281; Mehvar,R., J. Pharm Pharm Sci., 3(1):125-136 (2000) which are incorporated byreference herein). The invention also includes biomaterials comprising asurface having one or more reactive azide or acetylene sites and one ormore of the azide- or acetylene-containing polymers of the inventioncoupled to the surface via the Huisgen [3+2]cycloaddition linkage.Biomaterials and other substances can also be coupled to the azide- oracetylene-activated polymer derivatives through a linkage other than theazide or acetylene linkage, such as through a linkage comprising acarboxylic acid, amine, alcohol or thiol moiety, to leave the azide oracetylene moiety available for subsequent reactions.

The invention includes a method of synthesizing the azide- andacetylene-containing polymers of the invention. In the case of theazide-containing PEG derivative, the azide can be bonded directly to acarbon atom of the polymer. Alternatively, the azide-containing PEGderivative can be prepared by attaching a linking agent that has theazide moiety at one terminus to a conventional activated polymer so thatthe resulting polymer has the azide moiety at its terminus. In the caseof the acetylene-containing PEG derivative, the acetylene can be bondeddirectly to a carbon atom of the polymer. Alternatively, theacetylene-containing PEG derivative can be prepared by attaching alinking agent that has the acetylene moiety at one terminus to aconventional activated polymer so that the resulting polymer has theacetylene moiety at its terminus.

More specifically, in the case of the azide-containing PEG derivative, awater soluble polymer having at least one active hydroxyl moietyundergoes a reaction to produce a substituted polymer having a morereactive moiety, such as a mesylate, tresylate, tosylate or halogenleaving group, thereon. The preparation and use of PEG derivativescontaining sulfonyl acid halides, halogen atoms and other leaving groupsare known to those of ordinary skill in the art. The resultingsubstituted polymer then undergoes a reaction to substitute for the morereactive moiety an azide moiety at the terminus of the polymer.Alternatively, a water soluble polymer having at least one activenucleophilic or electrophilic moiety undergoes a reaction with a linkingagent that has an azide at one terminus so that a covalent bond isformed between the PEG polymer and the linking agent and the azidemoiety is positioned at the terminus of the polymer. Nucleophilic andelectrophilic moieties, including amines, thiols, hydrazides,hydrazines, alcohols, carboxylates, aldehydes, ketones, thioesters andthe like, are known to those of ordinary skill in the art.

More specifically, in the case of the acetylene-containing PEGderivative, a water soluble polymer having at least one active hydroxylmoiety undergoes a reaction to displace a halogen or other activatedleaving group from a precursor that contains an acetylene moiety.Alternatively, a water soluble polymer having at least one activenucleophilic or electrophilic moiety undergoes a reaction with a linkingagent that has an acetylene at one terminus so that a covalent bond isformed between the PEG polymer and the linking agent and the acetylenemoiety is positioned at the terminus of the polymer. The use of halogenmoieties, activated leaving group, nucleophilic and electrophilicmoieties in the context of organic synthesis and the preparation and useof PEG derivatives is well established to practitioners in the art.

The invention also provides a method for the selective modification ofproteins to add other substances to the modified protein, including butnot limited to water soluble polymers such as PEG and PEG derivativescontaining an azide or acetylene moiety. The azide- andacetylene-containing PEG derivatives can be used to modify theproperties of surfaces and molecules where biocompatibility, stability,solubility and lack of immunogenicity are important, while at the sametime providing a more selective means of attaching the PEG derivativesto proteins than was previously known in the art.

II. Growth Hormone Supergene Family

The following proteins include those encoded by genes of the growthhormone (GH) supergene family (Bazan, F., Immunology Today 11: 350-354(1990); Bazan, J. F. Science 257: 410-413 (1992); Mott, H. R. andCampbell, I. D., Current Opinion in Structural Biology 5: 114-121(1995); Silvennoinen, O. and Ihle, J. N., SIGNALLING BY THEHEMATOPOIETIC CYTOKINE RECEPTORS (1996)): growth hormone, prolactin,placental lactogen, erythropoietin (EPO), thrombopoietin (TPO),interleukin-2 (IL-2), IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11,IL-12 (p35 subunit), IL-13, IL-15, oncostatin M, ciliary neurotrophicfactor (CNTF), leukemia inhibitory factor (LIF), alpha interferon, betainterferon, epsilon interferon, gamma interferon, omega interferon, tauinterferon, granulocyte-colony stimulating factor (G-CSF),granulocyte-macrophage colony stimulating factor (GM-CSF), macrophagecolony stimulating factor (M-CSF) and cardiotrophin-1 (CT-1) (“the GHsupergene family”). It is anticipated that additional members of thisgene family will be identified in the future through gene cloning andsequencing. Members of the GH supergene family have similar secondaryand tertiary structures, despite the fact that they generally havelimited amino acid or DNA sequence identity. The shared structuralfeatures allow new members of the gene family to be readily identifiedand the non-natural amino acid methods and compositions described hereinsimilarly applied. Given the extent of structural homology among themembers of the GH supergene family, non-naturally encoded amino acidsmay be incorporated into any members of the GH supergene family usingthe present invention. Each member of this family of proteins comprisesa four helical bundle, the general structure of which is shown inFIG. 1. The general structures of family members hGH, EPO, IFNα-2, andG-CSF are shown in FIGS. 2, 3, 4, and 5, respectively.

Structures of a number of cytokines, including G-CSF (Zink et al., FEBSLett. 314:435 (1992); Zink et al., Biochemistry 33:8453 (1994); Hill etal., Proc. Natl. Acad. Sci. USA 90:5167 (1993), GM-CSF (Diederichs, K.,et al. Science 154: 1779-1782 (1991); Walter et al., J. Mol. Biol.224:1075-1085 (1992)), IL-2 (Bazan, J. F. and McKay, D. B. Science 257:410-413 (1992), IL-4 (Redfield et al., Biochemistry 30: 11029-11035(1991); Powers et al., Science 256:1673-1677 (1992)), and IL-5 (Milbumet al., Nature 363: 172-176 (1993)) have been determined by X-raydiffraction and NMR studies and show striking conservation with the GHstructure, despite a lack of significant primary sequence homology. IFNis considered to be a member of this family based upon modeling andother studies (Lee et al., J. Interferon Cytokine Res. 15:341 (1995);Murgolo et al., Proteins 17:62 (1993); Radhakrishnan et al., Structure4:1453 (1996); Klaus et al., J. Mol. Biol. 274:661 (1997)). EPO isconsidered to be a member of this family based upon modeling andmutagenesis studies (Boissel et al., J. Biol. Chem. 268: 15983-15993(1993); Wen et al., J. Biol. Chem. 269: 22839-22846 (1994)). All of theabove cytokines and growth factors are now considered to comprise onelarge gene family.

In addition to sharing similar secondary and tertiary structures,members of this family share the property that they must oligomerizecell surface receptors to activate intracellular signaling pathways.Some GH family members, including but not limited to; GH and EPO, bind asingle type of receptor and cause it to form homodimers. Other familymembers, including but not limited to, IL-2, IL-4, and IL-6, bind morethan one type of receptor and cause the receptors to form heterodimersor higher order aggregates (Davis et al., (1993), Science 260:1805-1808; Paonessa et al., (1995), EMBO J. 14: 1942-1951; Mott andCampbell, Current Opinion in Structural Biology 5: 114-121 (1995)).Mutagenesis studies have shown that, like GH, these other cytokines andgrowth factors contain multiple receptor binding sites, typically two,and bind their cognate receptors sequentially (Mott and Campbell,Current Opinion in Structural Biology 5: 114-121 (1995); Matthews etal., (1996) Proc. Natl. Acad. Sci. USA 93: 9471-9476). Like GH, theprimary receptor binding sites for these other family members occurprimarily in the four alpha helices and the A-B loop. The specific aminoacids in the helical bundles that participate in receptor binding differamongst the family members. Most of the cell surface receptors thatinteract with members of the GH supergene family are structurallyrelated and comprise a second large multi-gene family. See, e.g. U.S.Pat. No. 6,608,183, which is incorporated by reference herein.

A general conclusion reached from mutational studies of various membersof the GH supergene family is that the loops joining the alpha helicesgenerally tend to not be involved in receptor binding. In particular theshort B-C loop appears to be non-essential for receptor binding in most,if not all, family members. For this reason, the B-C loop may besubstituted with non-naturally encoded amino acids as described hereinin members of the GH supergene family. The A-B loop, the C-D loop (andD-E loop of interferon/IL-10-like members of the GH superfamily) mayalso be substituted with a non-naturally-occurring amino acid. Aminoacids proximal to helix A and distal to the final helix also tend not tobe involved in receptor binding and also may be sites for introducingnon-naturally-occurring amino acids. In some embodiments, anon-naturally encoded amino acid is substituted at any position within aloop structure, including but not limited to, the first 1, 2, 3, 4, 5,6, 7, or more amino acids of the A-B, B-C, C-D or D-E loop. In someembodiments, one or more non-naturally encoded amino acids aresubstituted within the last 1, 2, 3, 4, 5, 6, 7, or more amino acids ofthe A-B, B-C, C-D or D-E loop.

Certain members of the GH family, including but not limited to, EPO,IL-2, IL-3, IL-4, IL-6, G-CSF, GM-CSF, TPO, IL-10, IL-12 p35, IL-13,IL-15 and beta interferon contain N-linked and/or O-linked sugars. Theglycosylation sites in the proteins occur almost exclusively in the loopregions and not in the alpha helical bundles. Because the loop regionsgenerally are not involved in receptor binding and because they aresites for the covalent attachment of sugar groups, they may be usefulsites for introducing non-naturally-occurring amino acid substitutionsinto the proteins. Amino acids that comprise the N- and O-linkedglycosylation sites in the proteins may be sites fornon-naturally-occurring amino acid substitutions because these aminoacids are surface-exposed. Therefore, the natural protein can toleratebulky sugar groups attached to the proteins at these sites and theglycosylation sites tend to be located away from the receptor bindingsites.

Additional members of the GH supergene family are likely to bediscovered in the future. New members of the GH supergene family can beidentified through computer-aided secondary and tertiary structureanalyses of the predicted protein sequences, and by selection techniquesdesigned to identify molecules that bind to a particular target. Membersof the GH supergene family typically possess four or five amphipathichelices joined by non-helical amino acids (the loop regions). Theproteins may contain a hydrophobic signal sequence at their N-terminusto promote secretion from the cell. Such later discovered members of theGH supergene family also are included within this invention. A relatedapplication is International Patent Application entitled “Modified FourHelical Bundle Polypeptides and Their Uses” published as WO 05/074650 onAug. 18, 2005, which is incorporated by reference herein.

Thus, the description of the growth hormone supergene family is providedfor illustrative purposes and by way of example only and not as a limiton the scope of the methods, compositions, strategies and techniquesdescribed herein. Further, reference to GH polypeptides in thisapplication is intended to use the generic term as an example of anymember of the GH supergene family. Thus, it is understood that themodifications and chemistries described herein with reference to hGHpolypeptides or protein can be equally applied to any member of the GHsupergene family, including those specifically listed herein.

III. General Recombinant Nucleic Acid Methods for Use with the Invention

In numerous embodiments of the present invention, nucleic acids encodinga GH, e.g., hGH polypeptide of interest will be isolated, cloned andoften altered using recombinant methods. Such embodiments are used,including but not limited to, for protein expression or during thegeneration of variants, derivatives, expression cassettes, or othersequences derived from a GH, e.g., hGH polypeptide. In some embodiments,the sequences encoding the polypeptides of the invention are operablylinked to a heterologous promoter. Isolation of hGH and production of GHin host cells are described in, e.g., U.S. Pat. Nos. 4,601,980,4,604,359, 4,634,677, 4,658,021, 4,898,830, 5,424,199, 5,795,745,5,854,026, 5,849,535; 6,004,931; 6,022,711; 6,143,523 and 6,608,183,which are incorporated by reference herein.

A nucleotide sequence encoding a hGH polypeptide comprising anon-naturally encoded amino acid may be synthesized on the basis of theamino acid sequence of the parent polypeptide, including but not limitedto, having the amino acid sequence shown in SEQ ID NO: 2 (hGH) and thenchanging the nucleotide sequence so as to effect introduction (i.e.,incorporation or substitution) or removal (i.e., deletion orsubstitution) of the relevant amino acid residue(s). The nucleotidesequence may be conveniently modified by site-directed mutagenesis inaccordance with conventional methods. Alternatively, the nucleotidesequence may be prepared by chemical synthesis, including but notlimited to, by using an oligonucleotide synthesizer, whereinoligonucleotides are designed based on the amino acid sequence of thedesired polypeptide, and preferably selecting those codons that arefavored in the host cell in which the recombinant polypeptide will beproduced. For example, several small oligonucleotides coding forportions of the desired polypeptide may be synthesized and assembled byPCR, ligation or ligation chain reaction. See, e.g., Barany, et al.,Proc. Natl. Acad. Sci. 88: 189-193 (1991); U.S. Pat. No. 6,521,427 whichare incorporated by reference herein.

This invention utilizes routine techniques in the field of recombinantgenetics. Basic texts disclosing the general methods of use in thisinvention include Sambrook et al., Molecular Cloning, A LaboratoryManual (3rd ed. 2001); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994)).

General texts which describe molecular biological techniques includeBerger and Kimmel, Guide to Molecular Cloning Techniques, Methods inEnzymology volume 152 Academic Press, Inc., San Diego, Calif. (Berger);Sambrook et al., Molecular Cloning—A Laboratory Manual (2nd Ed.) Vol.1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989(“Sambrook”) and Current Protocols in Molecular Biology, F. M. Ausubelet al., eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (supplementedthrough 1999) (“Ausubel”)). These texts describe mutagenesis, the use ofvectors, promoters and many other relevant topics related to, includingbut not limited to, the generation of genes or polynucleotides thatinclude selector codons for production of proteins that includeunnatural amino acids, orthogonal tRNAs, orthogonal synthetases, andpairs thereof.

Various types of mutagenesis are used in the invention for a variety ofpurposes, including but not limited to, to produce novel synthetases ortRNAs, to mutate tRNA molecules, to mutate polynucleotides encodingsynthetases, to produce libraries of tRNAs, to produce libraries ofsynthetases, to produce selector codons, to insert selector codons thatencode unnatural amino acids in a protein or polypeptide of interest.They include but are not limited to site-directed, random pointmutagenesis, homologous recombination, DNA shuffling or other recursivemutagenesis methods, chimeric construction, mutagenesis using uracilcontaining templates, oligonucleotide-directed mutagenesis,phosphorothioate-modified DNA mutagenesis, mutagenesis using gappedduplex DNA or the like, or any combination thereof. Additional suitablemethods include point mismatch repair, mutagenesis usingrepair-deficient host strains, restriction-selection andrestriction-purification, deletion mutagenesis, mutagenesis by totalgene synthesis, double-strand break repair, and the like. Mutagenesis,including but not limited to, involving chimeric constructs, are alsoincluded in the present invention. In one embodiment, mutagenesis can beguided by known information of the naturally occurring molecule oraltered or mutated naturally occurring molecule, including but notlimited to, sequence, sequence comparisons, physical properties,secondary, tertiary, or quaternary structure, crystal structure or thelike.

The texts and examples found herein describe these procedures.Additional information is found in the following publications andreferences cited within: Ling et al., Approaches to DNA mutagenesis: anoverview, Anal Biochem. 254(2): 157-178 (1997); Dale et al.,Oligonucleotide-directed random mutagenesis using the phosphorothioatemethod, Methods Mol. Biol. 57:369-374 (1996); Smith, In vitromutagenesis, Ann. Rev. Genet. 19:423-462 (1985); Botstein & Shortle,Strategies and applications of in vitro mutagenesis, Science229:1193-1201 (1985); Carter, Site-directed mutagenesis, Biochem. J.237:1-7 (1986); Kunkel, The efficiency of oligonucleotide directedmutagenesis, in Nucleic Acids & Molecular Biology (Eckstein, F. andLilley, D. M. J. eds., Springer Verlag, Berlin) (1987); Kunkel, Rapidand efficient site-specific mutagenesis without phenotypic selection,Proc. Natl. Acad. Sci. USA 82:488-492 (1985); Kunkel et al., Rapid andefficient site-specific mutagenesis without phenotypic selection,Methods in Enzymol. 154, 367-382 (1987); Bass et al., Mutant Trprepressors with new DNA-binding specificities, Science 242:240-245(1988); Zoller & Smith, Oligonucleotide-directed mutagenesis usingM13-derived vectors: an efficient and general procedure for theproduction of point mutations in any DNA fragment, Nucleic Acids Res.10:6487-6500 (1982); Zoller & Smith, Oligonucleotide-directedmutagenesis of DNA fragments cloned into M13 vectors, Methods inEnzymol. 100:468-500 (1983); Zoller & Smith, Oligonucleotide-directedmutagenesis: a simple method using two oligonucleotide primers and asingle-stranded DNA template, Methods in Enzymol. 154:329-350 (1987);Taylor et al., The use of phosphorothioate-modified DNA in restrictionenzyme reactions to prepare nicked DNA, Nucl. Acids Res. 13: 8749-8764(1985); Taylor et al., The rapid generation of oligonucleotide-directedmutations at high frequency using phosphorothioate-modified DNA, Nucl.Acids Res. 13: 8765-8785 (1985); Nakamaye & Eckstein, Inhibition ofrestriction endonuclease Nci I cleavage by phosphorothioate groups andits application to oligonucleotide-directed mutagenesis, Nucl. AcidsRes. 14: 9679-9698 (1986); Sayers et al., 5′-3 Exonucleases inphosphorothioate-based oligonucleotide-directed mutagenesis, Nucl. AcidsRes. 16:791-802 (1988); Sayers et al., Strand specific cleavage ofphosphorothioate-containing DNA by reaction with restrictionendonucleases in the presence of ethidium bromide, (1988) Nucl. AcidsRes. 16: 803-814; Kramer et al., The gapped duplex DNA approach tooligonucleotide-directed mutation construction, Nucl. Acids Res. 12:9441-9456 (1984); Kramer & Fritz Oligonucleotide-directed constructionof mutations via gapped duplex DNA, Methods in Enzymol. 154:350-367(1987); Kramer et al., Improved enzymatic in vitro reactions in thegapped duplex DNA approach to oligonucleotide-directed construction ofmutations, Nucl. Acids Res. 16: 7207 (1988); Fritz et al.,Oligonucleotide-directed construction of mutations: a gapped duplex DNAprocedure without enzymatic reactions in vitro, Nucl. Acids Res. 16:6987-6999 (1988); Kramer et al., Different base/base mismatches arecorrected with different efficiencies by the methyl-directed DNAmismatch-repair system of E. coli, Cell 38:879-887 (1984); Carter etal., Improved oligonucleotide site-directed mutagenesis using M13vectors, Nucl. Acids Res. 13: 4431-4443 (1985); Carter, Improvedoligonucleotide-directed mutagenesis using M13 vectors, Methods inEnzymol. 154: 382-403 (1987); Eghtedarzadeh & Henikoff, Use ofoligonucleotides to generate large deletions, Nucl. Acids Res. 14: 5115(1986); Wells et al., Importance of hydrogen-bond formation instabilizing the transition state of subtilisin, Phil. Trans. R. Soc.Lond. A 317: 415-423 (1986); Nambiar et al., Total synthesis and cloningof a gene coding for the ribonuclease S protein, Science 223: 1299-1301(1984); Sakmar and Khorana, Total synthesis and expression of a gene forthe alpha-subunit of bovine rod outer segment guanine nucleotide-bindingprotein (transducin), Nucl. Acids Res. 14: 6361-6372 (1988); Wells etal., Cassette mutagenesis: an efficient method for generation ofmultiple mutations at defined sites, Gene 34:315-323 (1985); Grundströmet al., Oligonucleotide-directed mutagenesis by microscale ‘shot-gun’gene synthesis, Nucl. Acids Res. 13: 3305-3316 (1985); Mandecki,Oligonucleotide-directed double-strand break repair in plasmids ofEscherichia coli: a method for site-specific mutagenesis, Proc. Natl.Acad. Sci. USA, 83:7177-7181 (1986); Arnold, Protein engineering forunusual environments, Current Opinion in Biotechnology 4:450-455 (1993);Sieber, et al., Nature Biotechnology, 19:456-460 (2001); W. P. C.Stemmer, Nature 370, 389-91 (1994); and, I. A. Lorimer, I. Pastan,Nucleic Acids Res. 23, 3067-8 (1995). Additional details on many of theabove methods can be found in Methods in Enzymology Volume 154, whichalso describes useful controls for trouble-shooting problems withvarious mutagenesis methods.

Oligonucleotides, e.g., for use in mutagenesis of the present invention,e.g., mutating libraries of synthetases, or altering tRNAs, aretypically synthesized chemically according to the solid phasephosphoramidite triester method described by Beaucage and Caruthers,Tetrahedron Letts. 22(20):1859-1862, (1981) e.g., using an automatedsynthesizer, as described in Needham-VanDevanter et al., Nucleic AcidsRes., 12:6159-6168 (1984).

The invention also relates to eukaryotic host cells, non-eukaryotic hostcells, and organisms for the in vivo incorporation of an unnatural aminoacid via orthogonal tRNA/RS pairs. Host cells are genetically engineered(including but not limited to, transformed, transduced or transfected)with the polynucleotides of the invention or constructs which include apolynucleotide of the invention, including but not limited to, a vectorof the invention, which can be, for example, a cloning vector or anexpression vector. For example, the coding regions for the orthogonaltRNA, the orthogonal tRNA synthetase, and the protein to be derivatizedare operably linked to gene expression control elements that arefunctional in the desired host cell. The vector can be, for example, inthe form of a plasmid, a cosmid, a phage, a bacterium, a virus, a nakedpolynucleotide, or a conjugated polynucleotide. The vectors areintroduced into cells and/or microorganisms by standard methodsincluding electroporation (Fromm et al., Proc. Natl. Acad. Sci. USA 82,5824 (1985)), infection by viral vectors, high velocity ballisticpenetration by small particles with the nucleic acid either within thematrix of small beads or particles, or on the surface (Klein et al.,Nature 327, 70-73 (1987), and/or the like.

The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for such activities as, for example, screeningsteps, activating promoters or selecting transformants. These cells canoptionally be cultured into transgenic organisms. Other usefulreferences, including but not limited to for cell isolation and culture(e.g., for subsequent nucleic acid isolation) include Freshney (1994)Culture of Animal Cells, a Manual of Basic Technique, third edition,Wiley-Liss, New York and the references cited therein; Payne et al.(1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley &Sons, Inc. New York, N.Y.; Gamborg and Phillips (eds.) (1995) PlantCell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual,Springer-Verlag (Berlin Heidelberg New York) and Atlas and Parks (eds.)The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, Fla.

Several well-known methods of introducing target nucleic acids intocells are available, any of which can be used in the invention. Theseinclude: fusion of the recipient cells with bacterial protoplastscontaining the DNA, electroporation, projectile bombardment, andinfection with viral vectors (discussed further, below), etc. Bacterialcells can be used to amplify the number of plasmids containing DNAconstructs of this invention. The bacteria are grown to log phase andthe plasmids within the bacteria can be isolated by a variety of methodsknown in the art (see, for instance, Sambrook). In addition, kits arecommercially available for the purification of plasmids from bacteria,(see, e.g., EasyPrep™, FlexiPrep™, both from Pharmacia Biotech;StrataClean™ from Stratagene; and, QIAprep™ from Qiagen). The isolatedand purified plasmids are then further manipulated to produce otherplasmids, used to transfect cells or incorporated into related vectorsto infect organisms. Typical vectors contain transcription andtranslation terminators, transcription and translation initiationsequences, and promoters useful for regulation of the expression of theparticular target nucleic acid. The vectors optionally comprise genericexpression cassettes containing at least one independent terminatorsequence, sequences permitting replication of the cassette ineukaryotes, or prokaryotes, or both, (including but not limited to,shuttle vectors) and selection markers for both prokaryotic andeukaryotic systems. Vectors are suitable for replication and integrationin prokaryotes, eukaryotes, or both. See, Gillam & Smith, Gene 8:81(1979); Roberts, et al., Nature, 328:731 (1987); Schneider, E., et al.,Protein Expr. Purif. 6(1):10-14 (1995); Ausubel, Sambrook, Berger (allsupra). A catalogue of bacteria and bacteriophages useful for cloning isprovided, e.g., by the ATCC, e.g., The ATCC Catalogue of Bacteria andBacteriophage (1992) Gherna et al. (eds) published by the ATCC.Additional basic procedures for sequencing, cloning and other aspects ofmolecular biology and underlying theoretical considerations are alsofound in Watson et al. (1992) Recombinant DNA Second Edition ScientificAmerican Books, NY. In addition, essentially any nucleic acid (andvirtually any labeled nucleic acid, whether standard or non-standard)can be custom or standard ordered from any of a variety of commercialsources, such as the Midland Certified Reagent Company (Midland, Tex.available on the World Wide Web at mcrc.com), The Great American GeneCompany (Ramona, Calif. available on the World Wide Web at genco.com),ExpressGen Inc. (Chicago, Ill. available on the World Wide Web atexpressgen.com), Operon Technologies Inc. (Alameda, Calif.) and manyothers.

(a) Selector Codons

Selector codons of the invention expand the genetic codon framework ofprotein biosynthetic machinery. For example, a selector codon includes,but is not limited to, a unique three base codon, a nonsense codon, suchas a stop codon, including but not limited to, an amber codon (UAG), anochre codon, or an opal codon (UGA), an unnatural codon, a four or morebase codon, a rare codon, or the like. It is readily apparent to thoseof ordinary skill in the art that there is a wide range in the number ofselector codons that can be introduced into a desired gene orpolynucleotide, including but not limited to, one or more, two or more,three or more, 4, 5, 6, 7, 8, 9, 10 or more in a single polynucleotideencoding at least a portion of the hGH polypeptide.

In one embodiment, the methods involve the use of a selector codon thatis a stop codon for the incorporation of one or more unnatural aminoacids in vivo in a cell. For example, an O-tRNA is produced thatrecognizes the stop codon, including but not limited to, UAG, and isaminoacylated by an O—RS with a desired unnatural amino acid. ThisO-tRNA is not recognized by the naturally occurring host'saminoacyl-tRNA synthetases. Conventional site-directed mutagenesis canbe used to introduce the stop codon, including but not limited to, TAG,at the site of interest in a polypeptide of interest. See, e.g., Sayers,J. R., et al. (1988), 5′-3′ Exonucleases in phosphorothioate-basedoligonucleotide-directed mutagenesis. Nucleic Acids Res, 16:791-802.When the O—RS, O-tRNA and the nucleic acid that encodes the polypeptideof interest are combined in vivo, the unnatural amino acid isincorporated in response to the UAG codon to give a polypeptidecontaining the unnatural amino acid at the specified position.

The incorporation of unnatural amino acids in vivo can be done withoutsignificant perturbation of the eukaryotic host cell. For example,because the suppression efficiency for the UAG codon depends upon thecompetition between the O-tRNA, including but not limited to, the ambersuppressor tRNA, and a eukaryotic release factor (including but notlimited to, eRF) (which binds to a stop codon and initiates release ofthe growing peptide from the ribosome), the suppression efficiency canbe modulated by, including but not limited to, increasing the expressionlevel of O-tRNA, and/or the suppressor tRNA.

Unnatural amino acids can also be encoded with rare codons. For example,when the arginine concentration in an in vitro protein synthesisreaction is reduced, the rare arginine codon, AGG, has proven to beefficient for insertion of Ala by a synthetic tRNA acylated withalanine. See, e.g., Ma et al., Biochemistry, 32:7939 (1993). In thiscase, the synthetic tRNA competes with the naturally occurring tRNAArg,which exists as a minor species in Escherichia coli. Some organisms donot use all triplet codons. An unassigned codon AGA in Micrococcusluteus has been utilized for insertion of amino acids in an in vitrotranscription/translation extract. See, e.g., Kowal and Oliver, Nucl.Acid. Res., 25:4685 (1997). Components of the present invention can begenerated to use these rare codons in vivo.

Selector codons also comprise extended codons, including but not limitedto, four or more base codons, such as, four, five, six or more basecodons. Examples of four base codons include, but are not limited to,AGGA, CUAG, UAGA, CCCU and the like. Examples of five base codonsinclude, but are not limited to, AGGAC, CCCCU, CCCUC, CUAGA, CUACU,UAGGC and the like. A feature of the invention includes using extendedcodons based on frameshift suppression. Four or more base codons caninsert, including but not limited to, one or multiple unnatural aminoacids into the same protein. For example, in the presence of mutatedO-tRNAs, including but not limited to, a special frameshift suppressortRNAs, with anticodon loops, for example, with at least 8-10 ntanticodon loops, the four or more base codon is read as single aminoacid. In other embodiments, the anticodon loops can decode, includingbut not limited to, at least a four-base codon, at least a five-basecodon, or at least a six-base codon or more. Since there are 256possible four-base codons, multiple unnatural amino acids can be encodedin the same cell using a four or more base codon. See, Anderson et al.,(2002) Exploring the Limits of Codon and Anticodon Size, Chemistry andBiology, 9:237-244; Magliery, (2001) Expanding the Genetic CodeSelection of Efficient Suppressors of Four-base Codons andIdentification of “Shifty” Four-base Codons with a Library Approach inEscherichia coli, J. Mol. Biol. 307: 755-769.

For example, four-base codons have been used to incorporate unnaturalamino acids into proteins using in vitro biosynthetic methods. See,e.g., Ma et al., (1993) Biochemistry, 32:7939; and Hohsaka et al.,(1999) J. Am. Chem. Soc., 121:34. CGGG and AGGU were used tosimultaneously incorporate 2-naphthylalanine and an NBD derivative oflysine into streptavidin in vitro with two chemically acylatedframeshift suppressor tRNAs. See, e.g., Hohsaka et al., (1999) J. Am.Chem. Soc., 121:12194. In an in vivo study, Moore et al. examined theability of tRNALeu derivatives with NCUA anticodons to suppress UAGNcodons (N can be U, A, G, or C), and found that the quadruplet UAGA canbe decoded by a tRNALeu with a UCUA anticodon with an efficiency of 13to 26% with little decoding in the 0 or −1 frame. See, Moore et al.,(2000) J. Mol. Biol. 298:195. In one embodiment, extended codons basedon rare codons or nonsense codons can be used in the present invention,which can reduce missense readthrough and frameshift suppression atother unwanted sites.

For a given system, a selector codon can also include one of the naturalthree base codons, where the endogenous system does not use (or rarelyuses) the natural base codon. For example, this includes a system thatis lacking a tRNA that recognizes the natural three base codon, and/or asystem where the three base codon is a rare codon.

Selector codons optionally include unnatural base pairs. These unnaturalbase pairs further expand the existing genetic alphabet. One extra basepair increases the number of triplet codons from 64 to 125. Propertiesof third base pairs include stable and selective base pairing, efficientenzymatic incorporation into DNA with high fidelity by a polymerase, andthe efficient continued primer extension after synthesis of the nascentunnatural base pair. Descriptions of unnatural base pairs which can beadapted for methods and compositions include, e.g., Hirao, et al.,(2002) An unnatural base pair for incorporating amino acid analoguesinto protein, Nature Biotechnology, 20:177-182. See, also, Wu, Y., etal., (2002) J. Am. Chem. Soc. 124:14626-14630. Other relevantpublications are listed below.

For in vivo usage, the unnatural nucleoside is membrane permeable and isphosphorylated to form the corresponding triphosphate. In addition, theincreased genetic information is stable and not destroyed by cellularenzymes. Previous efforts by Benner and others took advantage ofhydrogen bonding patterns that are different from those in canonicalWatson-Crick pairs, the most noteworthy example of which is theiso-C:iso-G pair. See, e.g., Switzer et al., (1989) J. Am. Chem. Soc.,111:8322; and Piccirilli et al., (1990) Nature, 343:33; Kool, (2000)Curr. Opin. Chem. Biol., 4:602. These bases in general mispair to somedegree with natural bases and cannot be enzymatically replicated. Kooland co-workers demonstrated that hydrophobic packing interactionsbetween bases can replace hydrogen bonding to drive the formation ofbase pair. See, Kool, (2000) Curr. Opin. Chem. Biol., 4:602; and Guckianand Kool, (1998) Angew. Chem. Int. Ed. Engl., 36, 2825. In an effort todevelop an unnatural base pair satisfying all the above requirements,Schultz, Romesberg and co-workers have systematically synthesized andstudied a series of unnatural hydrophobic bases. A PICS:PICS self-pairis found to be more stable than natural base pairs, and can beefficiently incorporated into DNA by Klenow fragment of Escherichia coliDNA polymerase I (KF). See, e.g., McMinn et al., (1999) J. Am. Chem.Soc., 121:115.85-6; and Ogawa et al., (2000) J. Am. Chem. Soc.,122:3274. A 3MN:3MN self-pair can be synthesized by KF with efficiencyand selectivity sufficient for biological function. See, e.g., Ogawa etal., (2000) J. Am. Chem. Soc., 122:8803. However, both bases act as achain terminator for further replication. A mutant DNA polymerase hasbeen recently evolved that can be used to replicate the PICS self pair.In addition, a 7AI self pair can be replicated. See, e.g., Tae et al.,(2001) J. Am. Chem. Soc., 123:7439. A novel metallobase pair, Dipic:Py,has also been developed, which forms a stable pair upon binding Cu(II).See, Meggers et al., (2000) J. Am. Chem. Soc., 122:10714. Becauseextended codons and unnatural codons are intrinsically orthogonal tonatural codons, the methods of the invention can take advantage of thisproperty to generate orthogonal tRNAs for them.

A translational bypassing system can also be used to incorporate anunnatural amino acid in a desired polypeptide. In a translationalbypassing system, a large sequence is incorporated into a gene but isnot translated into protein. The sequence contains a structure thatserves as a cue to induce the ribosome to hop over the sequence andresume translation downstream of the insertion.

In certain embodiments, the protein or polypeptide of interest (orportion thereof) in the methods and/or compositions of the invention isencoded by a nucleic acid. Typically, the nucleic acid comprises atleast one selector codon, at least two selector codons, at least threeselector codons, at least four selector codons, at least five selectorcodons, at least six selector codons, at least seven selector codons, atleast eight selector codons, at least nine selector codons, ten or moreselector codons.

Genes coding for proteins or polypeptides of interest can be mutagenizedusing methods well-known to one of skill in the art and described hereinto include, for example, one or more selector codon for theincorporation of an unnatural amino acid. For example, a nucleic acidfor a protein of interest is mutagenized to include one or more selectorcodon, providing for the incorporation of one or more unnatural aminoacids. The invention includes any such variant, including but notlimited to, mutant, versions of any protein, for example, including atleast one unnatural amino acid. Similarly, the invention also includescorresponding nucleic acids, i.e., any nucleic acid with one or moreselector codon that encodes one or more unnatural amino acid.

Nucleic acid molecules encoding a protein of interest such as a hGHpolypeptide may be readily mutated to introduce a cysteine at anydesired position of the polypeptide. Cysteine is widely used tointroduce reactive molecules, water soluble polymers, proteins, or awide variety of other molecules, onto a protein of interest. Methodssuitable for the incorporation of cysteine into a desired position of apolypeptide are known to those of ordinary skill in the art, such asthose described in U.S. Pat. No. 6,608,183, which is incorporated byreference herein, and standard mutagenesis techniques.

IV. Non-Naturally Encoded Amino Acids

A very wide variety of non-naturally encoded amino acids are suitablefor use in the present invention. Any number of non-naturally encodedamino acids can be introduced into a GH, e.g., hGH polypeptide. Ingeneral, the introduced non-naturally encoded amino acids aresubstantially chemically inert toward the 20 common, genetically-encodedamino acids (i.e., alanine, arginine, asparagine, aspartic acid,cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine). In some embodiments, thenon-naturally encoded amino acids include side chain functional groupsthat react efficiently and selectively with functional groups not foundin the 20 common amino acids (including but not limited to, azido,ketone, aldehyde and aminooxy groups) to form stable conjugates. Forexample, a GH, e.g., hGH polypeptide that includes a non-naturallyencoded amino acid containing an azido functional group can be reactedwith a polymer (including but not limited to, poly(ethylene glycol) or,alternatively, a second polypeptide containing an alkyne moiety to forma stable conjugate resulting for the selective reaction of the azide andthe alkyne functional groups to form a Huisgen [3+2]cycloadditionproduct.

The generic structure of an alpha-amino acid is illustrated as follows(Formula I):

A non-naturally encoded amino acid is typically any structure having theabove-listed formula wherein the R group is any substituent other thanone used in the twenty natural amino acids, and may be suitable for usein the present invention. Because the non-naturally encoded amino acidsof the invention typically differ from the natural amino acids only inthe structure of the side chain, the non-naturally encoded amino acidsform amide bonds with other amino acids, including but not limited to,natural or non-naturally encoded, in the same manner in which they areformed in naturally occurring polypeptides. However, the non-naturallyencoded amino acids have side chain groups that distinguish them fromthe natural amino acids. For example, R optionally comprises an alkyl-,aryl-, acyl-, keto-, azido-, hydroxyl-, hydrazine, cyano-, halo-,hydrazide, alkenyl, alkynl, ether, thiol, seleno-, sulfonyl-, borate,boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine,aldehyde, ester, thioacid, hydroxylamine, amino group, or the like orany combination thereof. Other non-naturally occurring amino acids ofinterest that may be suitable for use in the present invention include,but are not limited to, amino acids comprising a photoactivatablecross-linker, spin-labeled amino acids, fluorescent amino acids, metalbinding amino acids, metal-containing amino acids, radioactive aminoacids, amino acids with novel functional groups, amino acids thatcovalently or noncovalently interact with other molecules, photocagedand/or photoisomerizable amino acids, amino acids comprising biotin or abiotin analogue, glycosylated amino acids such as a sugar substitutedserine, other carbohydrate modified amino acids, keto-containing aminoacids, amino acids comprising polyethylene glycol or polyether, heavyatom substituted amino acids, chemically cleavable and/or photocleavableamino acids, amino acids with an elongated side chains as compared tonatural amino acids, including but not limited to, polyethers or longchain hydrocarbons, including but not limited to, greater than about 5or greater than about 10 carbons, carbon-linked sugar-containing aminoacids, redox-active amino acids, amino thioacid containing amino acids,and amino acids comprising one or more toxic moiety.

Exemplary non-naturally encoded amino acids that may be suitable for usein the present invention and that are useful for reactions with watersoluble polymers include, but are not limited to, those with carbonyl,aminooxy, hydrazine, hydrazide, semicarbazide, azide and alkyne reactivegroups. In some embodiments, non-naturally encoded amino acids comprisea saccharide moiety. Examples of such amino acids includeN-acetyl-L-glucosaminyl-L-serine, N-acetyl-L-galactosaminyl-L-serine,N-acetyl-L-glucosaminyl-L-threonine,N-acetyl-L-glucosaminyl-L-asparagine and O-mannosaminyl-L-serine.Examples of such amino acids also include examples where thenaturally-occurring N- or O-linkage between the amino acid and thesaccharide is replaced by a covalent linkage not commonly found innature—including but not limited to, an alkene, an oxime, a thioether,an amide and the like. Examples of such amino acids also includesaccharides that are not commonly found in naturally-occurring proteinssuch as 2-deoxy-glucose, 2-deoxygalactose and the like.

Many of the non-naturally encoded amino acids provided herein arecommercially available, e.g., from Sigma-Aldrich (St. Louis, Mo., USA),Novabiochem (a division of EMD Biosciences, Darmstadt, Germany), orPeptech (Burlington, Mass., USA). Those that are not commerciallyavailable are optionally synthesized as provided herein or usingstandard methods known to those of ordinary skill in the art. Fororganic synthesis techniques, see, e.g., Organic Chemistry by Fessendonand Fessendon, (1982, Second Edition, Willard Grant Press, BostonMass.); Advanced Organic Chemistry by March (Third Edition, 1985, Wileyand Sons, New York); and Advanced Organic Chemistry by Carey andSundberg (Third Edition, Parts A and B, 1990, Plenum Press, New York).See, also, U.S. Patent Application Publications 2003/0082575 and2003/0108885, which are incorporated by reference herein. In addition tounnatural amino acids that contain novel side chains, unnatural aminoacids that may be suitable for use in the present invention alsooptionally comprise modified backbone structures, including but notlimited to, as illustrated by the structures of Formula II and III:

wherein Z typically comprises OH, NH₂, SH, NH—R′, or S—R′; X and Y,which can be the same or different, typically comprise S or O, and R andR′, which are optionally the same or different, are typically selectedfrom the same list of constituents for the R group described above forthe unnatural amino acids having Formula I as well as hydrogen. Forexample, unnatural amino acids of the invention optionally comprisesubstitutions in the amino or carboxyl group as illustrated by FormulasII and III. Unnatural amino acids of this type include, but are notlimited to, α-hydroxy acids, α-thioacids, α-aminothiocarboxylates,including but not limited to, with side chains corresponding to thecommon twenty natural amino acids or unnatural side chains. In addition,substitutions at the α-carbon optionally include, but are not limitedto, L, D, or α-α-disubstituted amino acids such as D-glutamate,D-alanine, D-methyl-O-tyrosine, aminobutyric acid, and the like. Otherstructural alternatives include cyclic amino acids, such as prolineanalogues as well as 3, 4, 6, 7, 8, and 9 membered ring prolineanalogues, β and γ amino acids such as substituted β-alanine and γ-aminobutyric acid.

Many unnatural amino acids are based on natural amino acids, such astyrosine, glutamine, phenylalanine, and the like, and are suitable foruse in the present invention. Tyrosine analogs include, but are notlimited to, para-substituted tyrosines, ortho-substituted tyrosines, andmeta substituted tyrosines, where the substituted tyrosine comprises,including but not limited to, a keto group (including but not limitedto, an acetyl group), a benzoyl group, an amino group, a hydrazine, anhydroxyamine, a thiol group, a carboxy group, an isopropyl group, amethyl group, a C₆-C₂₀ straight chain or branched hydrocarbon, asaturated or unsaturated hydrocarbon, an O-methyl group, a polyethergroup, a nitro group, an alkynyl group or the like. In addition,multiply substituted aryl rings are also contemplated. Glutamine analogsthat may be suitable for use in the present invention include, but arenot limited to, α-hydroxy derivatives, γ-substituted derivatives, cyclicderivatives, and amide substituted glutamine derivatives. Examplephenylalanine analogs that may be suitable for use in the presentinvention include, but are not limited to, para-substitutedphenylalanines, ortho-substituted phenyalanines, and meta-substitutedphenylalanines, where the substituent comprises, including but notlimited to, a hydroxy group, a methoxy group, a methyl group, an allylgroup, an aldehyde, an azido, an iodo, a bromo, a keto group (includingbut not limited to, an acetyl group), a benzoyl, an alkynyl group, orthe like. Specific examples of unnatural amino acids that may besuitable for use in the present invention include, but are not limitedto, a p-acetyl-L-phenylalanine, an O-methyl-L-tyrosine, anL-3-(2-naphthyl)alanine, a 3-methyl-phenylalanine, anO-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, atri-O-acetyl-GlcNAcβ-serine, an L-Dopa, a fluorinated phenylalanine, anisopropyl-L-phenylalanine, a p-azido-L-phenylalanine, ap-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine,a phosphonoserine, a phosphonotyrosine, a p-iodo-phenylalanine, ap-bromophenylalanine, a p-amino-L-phenylalanine, anisopropyl-L-phenylalanine, and a p-propargyloxy-phenylalanine, and thelike. Examples of structures of a variety of unnatural amino acids thatmay be suitable for use in the present invention are provided in, forexample, WO 2002/085923 entitled “In vivo incorporation of unnaturalamino acids.” See also Kiick et al., (2002) Incorporation of azides intorecombinant proteins for chemoselective modification by the Staudingerligation, PNAS 99:19-24, which is incorporated by reference herein, foradditional methionine analogs.

In one embodiment, compositions of a GH, e.g., hGH polypeptide thatinclude an unnatural amino acid (such as p-(propargyloxy)-phenyalanine)are provided. Various compositions comprisingp-(propargyloxy)-phenyalanine and, including but not limited to,proteins and/or cells, are also provided. In one aspect, a compositionthat includes the p-(propargyloxy)-phenyalanine unnatural amino acid,further includes an orthogonal tRNA. The unnatural amino acid can bebonded (including but not limited to, covalently) to the orthogonaltRNA, including but not limited to, covalently bonded to the orthogonaltRNA though an amino-acyl bond, covalently bonded to a 3′OH or a 2′OH ofa terminal ribose sugar of the orthogonal tRNA, etc.

The chemical moieties via unnatural amino acids that can be incorporatedinto proteins offer a variety of advantages and manipulations of theprotein. For example, the unique reactivity of a keto functional groupallows selective modification of proteins with any of a number ofhydrazine- or hydroxylamine-containing reagents in vitro and in vivo. Aheavy atom unnatural amino acid, for example, can be useful for phasingX-ray structure data. The site-specific introduction of heavy atomsusing unnatural amino acids also provides selectivity and flexibility inchoosing positions for heavy atoms. Photoreactive unnatural amino acids(including but not limited to, amino acids with benzophenone andarylazides (including but not limited to, phenylazide) side chains), forexample, allow for efficient in vivo and in vitro photocrosslinking ofprotein. Examples of photoreactive unnatural amino acids include, butare not limited to, p-azido-phenylalanine and p-benzoyl-phenylalanine.The protein with the photoreactive unnatural amino acids can then becrosslinked at will by excitation of the photoreactive group-providingtemporal control. In one example, the methyl group of an unnatural aminocan be substituted with an isotopically labeled, including but notlimited to, methyl group, as a probe of local structure and dynamics,including but not limited to, with the use of nuclear magnetic resonanceand vibrational spectroscopy. Alkynyl or azido functional groups, forexample, allow the selective modification of proteins with moleculesthrough a [3+2]cycloaddition reaction.

A non-natural amino acid incorporated into a polypeptide at the aminoterminus can be composed of an R group that is any substituent otherthan one used in the twenty natural amino acids and a 2^(nd) reactivegroup different from the NH₂ group normally present in α-amino acids(see Formula I). A similar non-natural amino acid can be incorporated atthe carboxyl terminus with a 2^(nd) reactive group different from theCOOH group normally present in α-amino acids (see Formula I).

The unnatural amino acids of the invention may be selected or designedto provide additional characteristics unavailable in the twenty naturalamino acids. For example, unnatural amino acid may be optionallydesigned or selected to modify the biological properties of a proteininto which they are incorporated. For example, the following propertiesmay be optionally modified by inclusion of an unnatural amino acid intoa protein: toxicity, biodistribution, solubility, stability, e.g.,thermal, hydrolytic, oxidative, resistance to enzymatic degradation, andthe like, facility of purification and processing, structuralproperties, spectroscopic properties, chemical and/or photochemicalproperties, catalytic activity, redox potential, half-life, ability toreact with other molecules, e.g., covalently or noncovalently, and thelike.

Structure and Synthesis of Non-Natural Amino Acids: Carbonyl.Carbonyl-Like, Masked Carbonyl, Protected Carbonyl Groups, andHydroxylamine Groups

In some embodiments the present invention provides a GH, e.g., hGH,linked to a water soluble polymer, e.g., a PEG, by an oxime bond.

Many types of non-naturally encoded amino acids are suitable forformation of oxime bonds. These include, but are not limited to,non-naturally encoded amino acids containing a carbonyl, dicarbonyl, orhydroxylamine group. Such amino acids are described in U.S. PatentApplication Nos. 60/638,418; 60/638,527; and 60/639,195, entitled“Compositions containing, methods involving, and uses of non-naturalamino acids and polypeptides,” filed Dec. 22, 2004, which areincorporated herein by reference in their entirety. Such amino acids arealso described in U.S. Patent Application Nos. 60/696,210; 60/696,302;and 60/696,068, entitled “Compositions containing, methods involving,and uses of non-natural amino acids and polypeptides,” filed Jul. 1,2005, which are incorporated herein by reference in their entirety.Non-naturally encoded amino acids are also described in U.S. patentapplication Ser. No. 10/126,931 filed on 19 Apr. 2002 and U.S. patentapplication Ser. No. 10/126,927, filed 19 Apr. 2002, which areincorporated by reference herein in their entirety.

Some embodiments of the invention utilize GH, e.g., hGH polypeptidesthat are substituted at one or more positions with apara-acetylphenylalanine amino acid. The synthesis ofp-acetyl-(+/−)-phenylalanine and m-acetyl-(+/−)-phenylalanine aredescribed in Zhang, Z., et al., Biochemistry 42: 6735-6746 (2003),incorporated by reference. Other carbonyl- or dicarbonyl-containingamino acids can be similarly prepared by one of ordinary skill in theart. Further, non-limiting exemplary syntheses of non-natural amino acidthat are included herein are presented in FIGS. 4, 24-34 and 36-39 ofU.S. patent application Ser. No. 10/126,931, which is incorporated byreference herein in its entirety.

Amino acids with an electrophilic reactive group allow for a variety ofreactions to link molecules via nucleophilic addition reactions amongothers. Such electrophilic reactive groups include a carbonyl group(including a keto group and a dicarbonyl group), a carbonyl-like group(which has reactivity similar to a carbonyl group (including a ketogroup and a dicarbonyl group) and is structurally similar to a carbonylgroup), a masked carbonyl group (which can be readily converted into acarbonyl group (including a keto group and a dicarbonyl group)), or aprotected carbonyl group (which has reactivity similar to a carbonylgroup (including a keto group and a dicarbonyl group) upondeprotection). Such amino acids include amino acids having the structureof Formula (IV):

wherein:A is optional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;B is optional, and when present is a linker selected from the groupconsisting of lower alkylene, substituted lower alkylene, loweralkenylene, substituted lower alkenylene, lower heteroalkylene,substituted lower heteroalkylene, —O—, —O-(alkylene or substitutedalkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,—C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;each R″ is independently H, alkyl, substituted alkyl, or a protectinggroup, or when more than one R″ group is present, two R″ optionally forma heterocycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;each of R₃ and R₄ is independently H, halogen, lower alkyl, orsubstituted lower alkyl, or R₃ and R₄ or two R₃ groups optionally form acycloalkyl or a heterocycloalkyl;or the -A-B-J-R groups together form a bicyclic or tricyclic cycloalkylor heterocycloalkyl comprising at least one carbonyl group, including adicarbonyl group, protected carbonyl group, including a protecteddicarbonyl group, or masked carbonyl group, including a maskeddicarbonyl group;or the -J-R group together forms a monocyclic or bicyclic cycloalkyl orheterocycloalkyl comprising at least one carbonyl group, including adicarbonyl group, protected carbonyl group, including a protecteddicarbonyl group, or masked carbonyl group, including a maskeddicarbonyl group;with a proviso that when A is phenylene and each R₃ is H, B is present;and that when A is —(CH₂)₄— and each R₃ is H, B is not —NHC(O)(CH₂CH₂)—;and that when A and B are absent and each R₃ is H, R is not methyl.

In addition, having the structure of Formula (V) are included:

wherein:A is optional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;B is optional, and when present is a linker selected from the groupconsisting of lower alkylene, substituted lower alkylene, loweralkenylene, substituted lower alkenylene, lower heteroalkylene,substituted lower heteroalkylene, —O—, —O-(alkylene or substitutedalkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,—C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;with a proviso that when A is phenylene, B is present; and that when Ais —(CH₂)₄—, B is not —NHC(O)(CH₂CH₂)—; and that when A and B areabsent, R is not methyl.

In addition, amino acids having the structure of Formula (VI) areincluded:

wherein:B is a linker selected from the group consisting of lower alkylene,substituted lower alkylene, lower alkenylene, substituted loweralkenylene, lower heteroalkylene, substituted lower heteroalkylene, —O—,—O-(alkylene or substituted alkylene)-, —S—, —S-(alkylene or substitutedalkylene)-, —S(O)_(k)— where k is 1, 2, or 3, —S(O)_(k)(alkylene orsubstituted alkylene)-, —C(O)—, —C(O)-(alkylene or substitutedalkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—,—NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,—CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,—CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene orsubstituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—,—N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—,—C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—,where each R′ is independently H, alkyl, or substituted alkyl;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;each R_(a) is independently selected from the group consisting of H,halogen, alkyl, substituted alkyl, —N(R′)₂, —C(O)_(k)R′ where k is 1, 2,or 3, —C(O)N(R′)₂, —OR′, and —S(O)_(k)R′, where each R′ is independentlyH, alkyl, or substituted alkyl.

In addition, the following amino acids are included:

compounds are optionally amino protected group, carboxyl protected or asalt thereof. In addition, any of the following non-natural amino acidsmay be incorporated into a non-natural amino acid polypeptide.

In addition, the following amino acids having the structure of Formula(VII) are included:

whereinB is optional, and when present is a linker selected from the groupconsisting of lower alkylene, substituted lower alkylene, loweralkenylene, substituted lower alkenylene, lower heteroalkylene,substituted lower heteroalkylene, —O—, —O-(alkylene or substitutedalkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,—C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;each R_(a) is independently selected from the group consisting of H,halogen, alkyl, substituted alkyl, —N(R′)₂, —C(O)_(k)R′ where k is 1, 2,or 3, —C(O)N(R′)₂, —OR′, and —S(O)_(k)R′, where each R′ is independentlyH, alkyl, or substituted alkyl; and n is 0 to 8; with a proviso thatwhen A is —CH₂)₄—, B is not —NHC(O)(CH₂CH₂)—.

In addition, the following amino acids are included:

wherein such compounds are optionally amino protected, optionallycarboxyl protected, optionally amino protected and carboxyl protected,or a salt thereof. In addition, these non-natural amino acids and any ofthe following non-natural amino acids may be incorporated into anon-natural amino acid polypeptide.

In addition, the following amino acids having the structure of Formula(VIII) are included:

wherein A is optional, and when present is lower alkylene, substitutedlower alkylene, lower cycloalkylene, substituted lower cycloalkylene,lower alkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;B is optional, and when present is a linker selected from the groupconsisting of lower alkylene, substituted lower alkylene, loweralkenylene, substituted lower alkenylene, lower heteroalkylene,substituted lower heteroalkylene, —O—, —O-(alkylene or substitutedalkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,—C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide.

In addition, the following amino acids having the structure of Formula(IX) are included:

B is optional, and when present is a linker selected from the groupconsisting of lower alkylene, substituted lower alkylene, loweralkenylene, substituted lower alkenylene, lower heteroalkylene,substituted lower heteroalkylene, —O—, —O-(alkylene or substitutedalkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,—C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;wherein each R_(a) is independently selected from the group consistingof H, halogen, alkyl, substituted alkyl, —N(R′)₂, —C(O)_(k)R′ where k is1, 2, or 3, —C(O)N(R′)₂, —OR′, and —S(O)_(k)R′, where each R′ isindependently H, alkyl, or substituted alkyl.

In addition, the following amino acids are included:

wherein such compounds are optionally amino protected, optionallycarboxyl protected, optionally amino protected and carboxyl protected,or a salt thereof. In addition, these non-natural amino acids and any ofthe following non-natural amino acids may be incorporated into anon-natural amino acid polypeptide.

In addition, the following amino acids having the structure of Formula(X) are included:

wherein B is optional, and when present is a linker selected from thegroup consisting of lower alkylene, substituted lower alkylene, loweralkenylene, substituted lower alkenylene, lower heteroalkylene,substituted lower heteroalkylene, —O—, —O-(alkylene or substitutedalkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,—C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;each R_(a) is independently selected from the group consisting of H,halogen, alkyl, substituted alkyl, —N(R′)₂, —C(O)_(k)R′ where k is 1, 2,or 3, —C(O)N(R′)₂, —OR′, and —S(O)_(k)R′, where each R′ is independentlyH, alkyl, or substituted alkyl; and n is 0 to 8.

In addition, the following amino acids are included:

wherein such compounds are optionally amino protected, optionallycarboxyl protected, optionally amino protected and carboxyl protected,or a salt thereof. In addition, these non-natural amino acids and any ofthe following non-natural amino acids may be incorporated into anon-natural amino acid polypeptide.

In addition to monocarbonyl structures, the non-natural amino acidsdescribed herein may include groups such as dicarbonyl, dicarbonyl like,masked dicarbonyl and protected dicarbonyl groups.

For example, the following amino acids having the structure of Formula(XI) are included:

wherein A is optional, and when present is lower alkylene, substitutedlower alkylene, lower cycloalkylene, substituted lower cycloalkylene,lower alkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;B is optional, and when present is a linker selected from the groupconsisting of lower alkylene, substituted lower alkylene, loweralkenylene, substituted lower alkenylene, lower heteroalkylene,substituted lower heteroalkylene, —O—, —O-(alkylene or substitutedalkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,—C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide.

In addition, the following amino acids having the structure of Formula(XII) are included:

B is optional, and when present is a linker selected from the groupconsisting of lower alkylene, substituted lower alkylene, loweralkenylene, substituted lower alkenylene, lower heteroalkylene,substituted lower heteroalkylene, —O—, —O-(alkylene or substitutedalkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,—C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;wherein each R_(a) is independently selected from the group consistingof H, halogen, alkyl, substituted alkyl, —N(R′)₂, —C(O)_(k)R′ where k is1, 2, or 3, —C(O)N(R′)₂, —OR′, and —S(O)_(k)R′, where each R′ isindependently H, alkyl, or substituted alkyl.

In addition, the following amino acids are included:

wherein such compounds are optionally amino protected, optionallycarboxyl protected, optionally amino protected and carboxyl protected,or a salt thereof. In addition, these non-natural amino acids and any ofthe following non-natural amino acids may be incorporated into anon-natural amino acid polypeptide.

In addition, the following amino acids having the structure of Formula(XIII) are included:

wherein B is optional, and when present is a linker selected from thegroup consisting of lower alkylene, substituted lower alkylene, loweralkenylene, substituted lower alkenylene, lower heteroalkylene,substituted lower heteroalkylene, —O—, —O-(alkylene or substitutedalkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,—C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;each R_(a) is independently selected from the group consisting of H,halogen, alkyl, substituted alkyl, —N(R′)₂, —C(O)_(k)R′ where k is 1, 2,or 3, —C(O)N(R′)₂, —OR′, and —S(O)_(k)R′, where each R′ is independentlyH, alkyl, or substituted alkyl; and n is 0 to 8.

In addition, the following amino acids are included:

wherein such compounds are optionally amino protected, optionallycarboxyl protected, optionally amino protected and carboxyl protected,or a salt thereof. In addition, these non-natural amino acids and any ofthe following non-natural amino acids may be incorporated into anon-natural amino acid polypeptide.

In addition, the following amino acids having the structure of Formula(XIV) are included:

wherein:A is optional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;X₁ is C, S, or S(O); and L is alkylene, substituted alkylene,N(R′)(alkylene) or N(R′)(substituted alkylene), where R′ is H, alkyl,substituted alkyl, cycloalkyl, or substituted cycloalkyl.

In addition, the following amino acids having the structure of Formula(XIV-A) are included:

wherein:A is optional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;L is alkylene, substituted alkylene, N(R′)(alkylene) orN(R′)(substituted alkylene), where R′ is H, alkyl, substituted alkyl,cycloalkyl, or substituted cycloalkyl.

In addition, the following amino acids having the structure of Formula(XIV-B) are included:

wherein:A is optional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;L is alkylene, substituted alkylene, N(R′)(alkylene) orN(R′)(substituted alkylene), where R′ is H, alkyl, substituted alkyl,cycloalkyl, or substituted cycloalkyl.

In addition, the following amino acids having the structure of Formula(XV) are included:

wherein:A is optional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;X₁ is C, S, or S(O); and n is 0, 1, 2, 3, 4, or 5; and each R⁸ and R⁹ oneach CR⁸R⁹ group is independently selected from the group consisting ofH, alkoxy, alkylamine, halogen, alkyl, aryl, or any R⁸ and R⁹ cantogether form ═O or a cycloalkyl, or any to adjacent R⁸ groups cantogether form a cycloalkyl.

In addition, the following amino acids having the structure of Formula(XV-A) are included:

wherein:A is optional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;n is 0, 1, 2, 3, 4, or 5; and each R⁸ and R⁹ on each CR⁸R⁹ group isindependently selected from the group consisting of H, alkoxy,alkylamine, halogen, alkyl, aryl, or any R⁸ and R⁹ can together form ═Oor a cycloalkyl, or any to adjacent R⁸ groups can together form acycloalkyl.

In addition, the following amino acids having the structure of Formula(XV-B) are included:

wherein:A is optional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;n is 0, 1, 2, 3, 4, or 5; and each R⁸ and R⁹ on each CR⁸R⁹ group isindependently selected from the group consisting of H, alkoxy,alkylamine, halogen, alkyl, aryl, or any R⁸ and R⁹ can together form ═Oor a cycloalkyl, or any to adjacent R⁸ groups can together form acycloalkyl.

In addition, the following amino acids having the structure of Formula(XVI) are included:

wherein:A is optional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;X₁ is C, S, or S(O); and L is alkylene, substituted alkylene,N(R′)(alkylene) or N(R′)(substituted alkylene), where R′ is H, alkyl,substituted alkyl, cycloalkyl, or substituted cycloalkyl.

In addition, the following amino acids having the structure of Formula(XVI-A) are included:

wherein:A is optional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;L is alkylene, substituted alkylene, N(R′)(alkylene) orN(R′)(substituted alkylene), where R′ is H, alkyl, substituted alkyl,cycloalkyl, or substituted cycloalkyl.

In addition, the following amino acids having the structure of Formula(XVI-B) are included:

wherein:A is optional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;L is alkylene, substituted alkylene, N(R′)(alkylene) orN(R′)(substituted alkylene), where R′ is H, alkyl, substituted alkyl,cycloalkyl, or substituted cycloalkyl.

In addition, amino acids having the structure of Formula (XVII) areincluded:

wherein:A is optional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;

where (a) indicates bonding to the A group and (b) indicates bonding torespective carbonyl groups, R₃ and R₄ are independently chosen from H,halogen, alkyl, substituted alkyl, cycloalkyl, or substitutedcycloalkyl, or R₃ and R₄ or two R₃ groups or two R₄ groups optionallyform a cycloalkyl or a heterocycloalkyl;R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substitutedcycloalkyl;T₃ is a bond, C(R)(R), O, or S, and R is H, halogen, alkyl, substitutedalkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide.

In addition, amino acids having the structure of Formula (XVIII) areincluded:

wherein:where (a) indicates bonding to the A group and (b) indicates bonding torespective carbonyl groups, R₃ and R₄ are independently chosen from H,halogen, alkyl, substituted alkyl, cycloalkyl, or substitutedcycloalkyl, or R₃ and R₄ or two R₃ groups or two R₄ groups optionallyform a cycloalkyl or a heterocycloalkyl;R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substitutedcycloalkyl;T₃ is a bond, C(R)(R), O, or S, and R is H, halogen, alkyl, substitutedalkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;each R_(a) is independently selected from the group consisting of H,halogen, alkyl, substituted alkyl, —N(R′)₂, —C(O)_(k)R′ where k is 1, 2,or 3, —C(O)N(R′)₂, —OR′, and —S(O)_(k)R′, where each R′ is independentlyH, alkyl, or substituted alkyl.

In addition, amino acids having the structure of Formula (XIX) areincluded:

wherein:R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substitutedcycloalkyl; andT₃ is O, or S.

In addition, amino acids having the structure of Formula (XX) areincluded:

wherein:R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substitutedcycloalkyl.

In addition, the following amino acids having structures of Formula(XXI) are included:

The synthesis of p-acetyl-(+/−)-phenylalanine andm-acetyl-(+/−)-phenylalanine is described in Zhang, Z., et al.,Biochemistry 42: 6735-6746 (2003), incorporated by reference. Othercarbonyl- or dicarbonyl-containing amino acids can be similarly preparedby one of ordinary skill in the art. Further, non-limiting exemplarysyntheses of non-natural amino acid that are include herein arepresented in FIGS. 4, 24-34 and 36-39.

In some embodiments, a polypeptide comprising a non-natural amino acidis chemically modified to generate a reactive carbonyl or dicarbonylfunctional group. For instance, an aldehyde functionality useful forconjugation reactions can be generated from a functionality havingadjacent amino and hydroxyl groups. Where the biologically activemolecule is a polypeptide, for example, an N-terminal serine orthreonine (which may be normally present or may be exposed via chemicalor enzymatic digestion) can be used to generate an aldehydefunctionality under mild oxidative cleavage conditions using periodate.See, e.g., Gaertner, et. al., Bioconjug. Chem. 3: 262-268 (1992);Geoghegan, K. & Stroh, J., Bioconjug. Chem. 3:138-146 (1992); Gaertneret al., J. Biol. Chem. 269:7224-7230 (1994). However, methods known inthe art are restricted to the amino acid at the N-terminus of thepeptide or protein.

In the present invention, a non-natural amino acid bearing adjacenthydroxyl and amino groups can be incorporated into the polypeptide as a“masked” aldehyde functionality. For example, 5-hydroxylysine bears ahydroxyl group adjacent to the epsilon amine. Reaction conditions forgenerating the aldehyde typically involve addition of molar excess ofsodium metaperiodate under mild conditions to avoid oxidation at othersites within the polypeptide. The pH of the oxidation reaction istypically about 7.0. A typical reaction involves the addition of about1.5 molar excess of sodium meta periodate to a buffered solution of thepolypeptide, followed by incubation for about 10 minutes in the dark.See, e.g. U.S. Pat. No. 6,423,685.

The carbonyl or dicarbonyl functionality can be reacted selectively witha hydroxylamine-containing reagent under mild conditions in aqueoussolution to form the corresponding oxime linkage that is stable underphysiological conditions. See, e.g., Jencks, W. P., J. Am. Chem. Soc.81, 475-481 (1959); Shao, J. and Tam, J. P., J. Am. Chem. Soc.117:3893-3899 (1995). Moreover, the unique reactivity of the carbonyl ordicarbonyl group allows for selective modification in the presence ofthe other amino acid side chains. See, e.g., Cornish, V. W., et al., J.Am. Chem. Soc. 118:8150-8151 (1996); Geoghegan, K. F. & Stroh, J. G.,Bioconjug. Chem. 3:138-146 (1992); Mahal, L. K., et al., Science276:1125-1128 (1997).

Structure and Synthesis of Non-Natural Amino Acids:Hydroxylamine-Containing Amino Acids

U.S. Provisional Patent Application No. 60/638,418 is incorporated byreference in its entirety. Thus, the disclosures provided in Section V(entitled “Non-natural Amino Acids”),

Part B (entitled “Structure and Synthesis of Non-Natural Amino Acids:Hydroxylamine-Containing Amino Acids”), in U.S. Provisional PatentApplication No. 60/638,418 apply fully to the methods, compositions(including Formulas I-XXXV, techniques and strategies for making,purifying, characterizing, and using non-natural amino acids,non-natural amino acid polypeptides and modified non-natural amino acidpolypeptides described herein to the same extent as if such disclosureswere fully presented herein.

Chemical Synthesis of Unnatural Amino Acids

Many of the unnatural amino acids suitable for use in the presentinvention are commercially available, e.g., from Sigma (USA) or Aldrich(Milwaukee, Wis., USA). Those that are not commercially available areoptionally synthesized as provided herein or as provided in variouspublications or using standard methods known to those of ordinary skillin the art. For organic synthesis techniques, see, e.g., OrganicChemistry by Fessendon and Fessendon, (1982, Second Edition, WillardGrant Press, Boston Mass.); Advanced Organic Chemistry by March (ThirdEdition, 1985, Wiley and Sons, New York); and Advanced Organic Chemistryby Carey and Sundberg (Third Edition, Parts A and B, 1990, Plenum Press,New York). Additional publications describing the synthesis of unnaturalamino acids include, e.g., WO 2002/085923 entitled “In vivoincorporation of Unnatural Amino Acids;” Matsoukas et al., (1995) J.Med. Chem., 38, 4660-4669; King, F. E. & Kidd, D. A. A. (1949) A NewSynthesis of Glutamine and of γ-Dipeptides of Glutamic Acid fromPhthylated Intermediates. J. Chem. Soc. 3315-3319; Friedman, O. M. &Chatterrji, R. (1959) Synthesis of Derivatives of Glutamine as ModelSubstrates for Anti-Tumor Agents. J. Arm. Chem. Soc. 81, 3750-3752;Craig, J. C. et al. (1988) Absolute Configuration of the Enantiomers of7-Chloro-4 [[4-(diethylamino)-1-methylbutyl]amino]quinoline(Chloroquine). J. Org. Chem. 53, 1167-1170; Azoulay, M., Vilmont, M. &Frappier, F. (1991) Glutamine analogues as Potential Antimalarials, Eur.J. Med. Chem. 26, 201-5; Koskinen, A. M. P. & Rapoport, H. (1989)Synthesis of 4-Substituted Prolines as Conformationally ConstrainedAmino Acid Analogues. J. Org. Chem. 54, 1859-1866; Christie, B. D. &Rapoport, H. (1985) Synthesis of Optically Pure Pipecolates fromL-Asparagine. Application to the Total Synthesis of (+)-Apovincaminethrough Amino Acid Decarbonylation and Iminium Ion Cyclization. J. Org.Chem. 50:1239-1246; Barton et al., (1987) Synthesis of Novelalpha-Amino-Acids and Derivatives Using Radical Chemistry: Synthesis ofL- and D-alpha-Amino-Adipic Acids, L-alpha-aminopimelic Acid andAppropriate Unsaturated Derivatives. Tetrahedron 43:4297-4308; and,Subasinghe et al., (1992) Quisqualic acid analogues: synthesis ofbeta-heterocyclic 2-aminopropanoic acid derivatives and their activityat a novel quisqualate-sensitized site. J. Med. Chem. 35:4602-7. Seealso, U.S. Patent Publication No. US 2004/0198637 entitled “ProteinArrays,” which is incorporated by reference herein.

A. Carbonyl Reactive Groups

Amino acids with a carbonyl reactive group allow for a variety ofreactions to link molecules (including but not limited to, PEG or otherwater soluble molecules) via nucleophilic addition or aldol condensationreactions among others.

Exemplary carbonyl-containing amino acids can be represented as follows:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl; R₂ is H, alkyl, aryl, substituted alkyl, andsubstituted aryl; and R₃ is H, an amino acid, a polypeptide, or an aminoterminus modification group, and R₄ is H, an amino acid, a polypeptide,or a carboxy terminus modification group. In some embodiments, n is 1,R₁ is phenyl and R₂ is a simple alkyl (i.e., methyl, ethyl, or propyl)and the ketone moiety is positioned in the para position relative to thealkyl side chain. In some embodiments, n is 1, R₁ is phenyl and R₂ is asimple alkyl (i.e., methyl, ethyl, or propyl) and the ketone moiety ispositioned in the meta position relative to the alkyl side chain.

The synthesis of p-acetyl-(+/−)-phenylalanine andm-acetyl-(+/−)-phenylalanine is described in Zhang, Z., et al.,Biochemistry 42: 6735-6746 (2003), which is incorporated by referenceherein. Other carbonyl-containing amino acids can be similarly preparedby one of ordinary skill in the art.

In some embodiments, a polypeptide comprising a non-naturally encodedamino acid is chemically modified to generate a reactive carbonylfunctional group. For instance, an aldehyde functionality useful forconjugation reactions can be generated from a functionality havingadjacent amino and hydroxyl groups. Where the biologically activemolecule is a polypeptide, for example, an N-terminal serine orthreonine (which may be normally present or may be exposed via chemicalor enzymatic digestion) can be used to generate an aldehydefunctionality under mild oxidative cleavage conditions using periodate.See, e.g., Gaertner, et al., Bioconjug. Chem. 3: 262-268 (1992);Geoghegan, K. & Stroh, J., Bioconjug. Chem. 3:138-146 (1992); Gaertneret al., J. Biol. Chem. 269:7224-7230 (1994). However, methods known inthe art are restricted to the amino acid at the N-terminus of thepeptide or protein.

In the present invention, a non-naturally encoded amino acid bearingadjacent hydroxyl and amino groups can be incorporated into thepolypeptide as a “masked” aldehyde functionality. For example,5-hydroxylysine bears a hydroxyl group adjacent to the epsilon amine.Reaction conditions for generating the aldehyde typically involveaddition of molar excess of sodium metaperiodate under mild conditionsto avoid oxidation at other sites within the polypeptide. The pH of theoxidation reaction is typically about 7.0. A typical reaction involvesthe addition of about 1.5 molar excess of sodium meta periodate to abuffered solution of the polypeptide, followed by incubation for about10 minutes in the dark. See, e.g. U.S. Pat. No. 6,423,685, which isincorporated by reference herein.

The carbonyl functionality can be reacted selectively with a hydrazine-,hydrazide-, hydroxylamine-, or semicarbazide-containing reagent undermild conditions in aqueous solution to form the corresponding hydrazone,oxime, or semicarbazone linkages, respectively, that are stable underphysiological conditions. See, e.g., Jencks, W. P., J. Am. Chem. Soc.81, 475-481 (1959); Shao, J. and Tam, J. P., J. Am. Chem. Soc.117:3893-3899 (1995). Moreover, the unique reactivity of the carbonylgroup allows for selective modification in the presence of the otheramino acid side chains. See, e.g., Cornish, V. W., et al., J. Am. Chem.Soc. 118:8150-8151 (1996); Geoghegan, K. F. & Stroh, J. G., Bioconjug.Chem. 3:138-146 (1992); Mahal, L. K., et al., Science 276:1125-1128(1997).

B. Hydrazine, Hydrazide or Semicarbazide Reactive Groups

Non-naturally encoded amino acids containing a nucleophilic group, suchas a hydrazine, hydrazide or semicarbazide, allow for reaction with avariety of electrophilic groups to form conjugates (including but notlimited to, with PEG or other water soluble polymers).

Exemplary hydrazine, hydrazide or semicarbazide-containing amino acidscan be represented as follows:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl or not present; X, is O, N, or S or not present; R₂ isH, an amino acid, a polypeptide, or an amino terminus modificationgroup, and R₃ is H, an amino acid, a polypeptide, or a carboxy terminusmodification group.

In some embodiments, n is 4, R₁ is not present, and X is N. In someembodiments, n is 2, R₁ is not present, and X is not present: In someembodiments, n is 1, R₁ is phenyl, X is O, and the oxygen atom ispositioned para to the alphatic group on the aryl ring.

Hydrazide-, hydrazine-, and semicarbazide-containing amino acids areavailable from commercial sources. For instance, L-glutamate-γ-hydrazideis available from Sigma Chemical (St. Louis, Mo.). Other amino acids notavailable commercially can be prepared by one of ordinary skill in theart. See, e.g., U.S. Pat. No. 6,281,211, which is incorporated byreference herein.

Polypeptides containing non-naturally encoded amino acids that bearhydrazide, hydrazine or semicarbazide functionalities can be reactedefficiently and selectively with a variety of molecules that containaldehydes or other functional groups with similar chemical reactivity.See, e.g., Shao, J. and Tam, J., J. Am. Chem. Soc. 117:3893-3899 (1995).The unique reactivity of hydrazide, hydrazine and semicarbazidefunctional groups makes them significantly more reactive towardaldehydes, ketones and other electrophilic groups as compared to thenucleophilic groups present on the 20 common amino acids (including butnot limited to, the hydroxyl group of serine or threonine or the aminogroups of lysine and the N-terminus).

C. Aminooxy-Containing Amino Acids

Non-naturally encoded amino acids containing an aminooxy (also called ahydroxylamine) group allow for reaction with a variety of electrophilicgroups to form conjugates (including but not limited to, with PEG orother water soluble polymers). Like hydrazines, hydrazides andsemicarbazides, the enhanced nucleophilicity of the aminooxy grouppermits it to react efficiently and selectively with a variety ofmolecules that contain aldehydes or other functional groups with similarchemical reactivity. See, e.g., Shao, J. and Tam, J., J. Am. Chem. Soc.117:3893-3899 (1995); H. Hang and C. Bertozzi, Acc. Chem. Res. 34:727-736 (2001).

Whereas the result of reaction with a hydrazine group is thecorresponding hydrazone, however, an oxime results generally from thereaction of an aminooxy group with a carbonyl-containing group such as aketone.

Exemplary amino acids containing aminooxy groups can be represented asfollows:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl or not present; X is O, N, S or not present; m is 0-10;Y═C(O) or not present; R₂ is H, an amino acid, a polypeptide, or anamino terminus modification group, and R₃ is H, an amino acid, apolypeptide, or a carboxy terminus modification group. In someembodiments, n is 1, R₁ is phenyl, X is O, m is 1, and Y is present. Insome embodiments, n is 2, R₁ and X are not present, m is 0, and Y is notpresent.

Aminooxy-containing amino acids can be prepared from readily availableamino acid precursors (homoserine, serine and threonine). See, e.g., M.Carrasco and R. Brown, J. Org. Chem. 68: 8853-8858 (2003). Certainaminooxy-containing amino acids, such as L-2-amino-4-(aminooxy)butyricacid), have been isolated from natural sources (Rosenthal, G, Life Sci.60: 1635-1641 (1997). Other aminooxy-containing amino acids can beprepared by one of ordinary skill in the art.

D. Azide and Alkyne Reactive Groups

The unique reactivity of azide and alkyne functional groups makes themextremely useful for the selective modification of polypeptides andother biological molecules. Organic azides, particularly alphaticazides, and alkynes are generally stable toward common reactive chemicalconditions. In particular, both the azide and the alkyne functionalgroups are inert toward the side chains (i.e., R groups) of the 20common amino acids found in naturally-occurring polypeptides. Whenbrought into close proximity, however, the “spring-loaded” nature of theazide and alkyne groups is revealed and they react selectively andefficiently via Huisgen [3+2]cycloaddition reaction to generate thecorresponding triazole. See, e.g., Chin J., et al., Science 301:964-7(2003); Wang, Q., et al., J. Am. Chem. Soc. 125, 3192-3193 (2003); Chin,J. W., et al., J. Am. Chem. Soc. 124:9026-9027 (2002).

Because the Huisgen cycloaddition reaction involves a selectivecycloaddition reaction (see, e.g., Padwa, A., in COMPREHENSIVE ORGANICSYNTHESIS, Vol. 4, (ed. Trost, B. M., 1991), p. 1069-1109; Huisgen, R.in 1,3-DIPOLAR CYCLOADDITION CHEMISTRY, (ed. Padwa, A., 1984), p. 1-176)rather than a nucleophilic substitution, the incorporation ofnon-naturally encoded amino acids bearing azide and alkyne-containingside chains permits the resultant polypeptides to be modifiedselectively at the position of the non-naturally encoded amino acid.Cycloaddition reaction involving azide or alkyne-containing GH, e.g.,hGH polypeptide can be carried out at room temperature under aqueousconditions by the addition of Cu(II) (including but not limited to, inthe form of a catalytic amount of CuSO₄) in the presence of a reducingagent for reducing Cu(II) to Cu(I), in situ, in catalytic amount. See,e.g., Wang, Q., et al., J. Am. Chem. Soc. 125, 3192-3193 (2003); Tomoe,C. W., et al., J. Org. Chem. 67:3057-3064 (2002); Rostovtsev, et al.,Angew. Chem. Int. Ed. 41:2596-2599 (2002). Exemplary reducing agentsinclude, including but not limited to, ascorbate, metallic copper,quinine, hydroquinone, vitamin K, glutathione, cysteine, Fe²⁺, Co²⁺, andan applied electric potential.

In some cases, where a Huisgen [3+2]cycloaddition reaction between anazide and an alkyne is desired, the GH, e.g., hGH polypeptide comprisesa non-naturally encoded amino acid comprising an alkyne moiety and thewater soluble polymer to be attached to the amino acid comprises anazide moiety. Alternatively, the converse reaction (i.e., with the azidemoiety on the amino acid and the alkyne moiety present on the watersoluble polymer) can also be performed.

The azide functional group can also be reacted selectively with a watersoluble polymer containing an aryl ester and appropriatelyfunctionalized with an aryl phosphine moiety to generate an amidelinkage. The aryl phosphine group reduces the azide in situ and theresulting amine then reacts efficiently with a proximal ester linkage togenerate the corresponding amide. See, e.g., E. Saxon and C. Bertozzi,Science 287, 2007-2010 (2000). The azide-containing amino acid can beeither an alkyl azide (including but not limited to,2-amino-6-azido-1-hexanoic acid) or an aryl azide(p-azido-phenylalanine).

Exemplary water soluble polymers containing an aryl ester and aphosphine moiety can be represented as follows:

wherein X can be O, N, S or not present, Ph is phenyl, W is a watersoluble polymer and R can be H, alkyl, aryl, substituted alkyl andsubstituted aryl groups. Exemplary R groups include but are not limitedto —CH₂, —C(CH₃)₃, —OR′, —NR′R″, —SR′, -halogen, —C(O)R′, —CONR′R″,—S(O)₂R′, —S(O)₂NR′R″, —CN and —NO₂. R′, R″, R′″ and R″″ eachindependently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, including but notlimited to, aryl substituted with 1-3 halogens, substituted orunsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups.When a compound of the invention includes more than one R group, forexample, each of the R groups is independently selected as are each R′,R″, R′″ and R″″ groups when more than one of these groups is present.When R′ and R″ are attached to the same nitrogen atom, they can becombined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.For example, —NR′R″ is meant to include, but not be limited to,1-pyrrolidinyl and 4-morpholinyl. From the above discussion ofsubstituents, one of skill in the art will understand that the term“alkyl” is meant to include groups including carbon atoms bound togroups other than hydrogen groups, such as haloalkyl (including but notlimited to, —CF₃ and —CH₂CF₃) and acyl (including but not limited to,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

The azide functional group can also be reacted selectively with a watersoluble polymer containing a thioester and appropriately functionalizedwith an aryl phosphine moiety to generate an amide linkage. The arylphosphine group reduces the azide in situ and the resulting amine thenreacts efficiently with the thioester linkage to generate thecorresponding amide. Exemplary water soluble polymers containing athioester and a phosphine moiety can be represented as follows:

wherein n is 1-10; X can be O, N, S or not present, Ph is phenyl, and Wis a water soluble polymer.

Exemplary alkyne-containing amino acids can be represented as follows:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl or not present; X is O, N, S or not present; m is 0-10,R₂ is H, an amino acid, a polypeptide, or an amino terminus modificationgroup, and R₃ is H, an amino acid, a polypeptide, or a carboxy terminusmodification group. In some embodiments, n is 1, R₁ is phenyl, X is notpresent, m is 0 and the acetylene moiety is positioned in the paraposition relative to the alkyl side chain. In some embodiments, n is 1,R₁ is phenyl, X is O, m is 1 and the propargyloxy group is positioned inthe para position relative to the alkyl side chain (i.e.,O-propargyl-tyrosine). In some embodiments, n is 1, R₁ and X are notpresent and m is 0 (i.e., proparylglycine).

Alkyne-containing amino acids are commercially available. For example,propargylglycine is commercially available from Peptech (Burlington,Mass.). Alternatively, alkyne-containing amino acids can be preparedaccording to standard methods. For instance, p-propargyloxyphenylalaninecan be synthesized, for example, as described in Deiters, A., et al., J.Am. Chem. Soc. 125: 11782-11783 (2003), and 4-alkynyl-L-phenylalaninecan be synthesized as described in Kayser, B., et al., Tetrahedron53(7): 2475-2484 (1997). Other alkyne-containing amino acids can beprepared by one of ordinary skill in the art.

Exemplary azide-containing amino acids can be represented as follows:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, substitutedaryl or not present; X is O, N, S or not present; m is 0-10; R₂ is H, anamino acid, a polypeptide, or an amino terminus modification group, andR₃ is H, an amino acid, a polypeptide, or a carboxy terminusmodification group. In some embodiments, n is 1, R₁ is phenyl, X is notpresent, m is 0 and the azide moiety is positioned para to the alkylside chain. In some embodiments, n is 0-4 and R₁ and X are not present,and m=0. In some embodiments, n is 1, R₁ is phenyl, X is O, m is 2 andthe β-azidoethoxy moiety is positioned in the para position relative tothe alkyl side chain.

Azide-containing amino acids are available from commercial sources. Forinstance, 4-azidophenylalanine can be obtained from Chem-ImpexInternational, Inc. (Wood Dale, Ill.). For those azide-containing aminoacids that are not commercially available, the azide group can beprepared relatively readily using standard methods known to those ofordinary skill in the art, including but not limited to, viadisplacement of a suitable leaving group (including but not limited to,halide, mesylate, tosylate) or via opening of a suitably protectedlactone. See, e.g., Advanced Organic Chemistry by March (Third Edition,1985, Wiley and Sons, New York).

E. Aminothiol Reactive Groups

The unique reactivity of beta-substituted aminothiol functional groupsmakes them extremely useful for the selective modification ofpolypeptides and other biological molecules that contain aldehyde groupsvia formation of the thiazolidine. See, e.g., J. Shao and J. Tam, J. Am.Chem. Soc. 1995, 117 (14) 3893-3899. In some embodiments,beta-substituted aminothiol amino acids can be incorporated into GH,e.g., hGH polypeptides and then reacted with water soluble polymerscomprising an aldehyde functionality. In some embodiments, a watersoluble polymer, drug conjugate or other payload can be coupled to a GH,e.g., hGH polypeptide comprising a beta-substituted aminothiol aminoacid via formation of the thiazolidine.

Cellular Uptake of Unnatural Amino Acids

Unnatural amino acid uptake by a cell is one issue that is typicallyconsidered when designing and selecting unnatural amino acids, includingbut not limited to, for incorporation into a protein. For example, thehigh charge density of α-amino acids suggests that these compounds areunlikely to be cell permeable. Natural amino acids are taken up into theeukaryotic cell via a collection of protein-based transport systems. Arapid screen can be done which assesses which unnatural amino acids, ifany, are taken up by cells. See, e.g., the toxicity assays in, e.g.,U.S. Patent Publication No. US 2004/0198637 entitled “Protein Arrays”which is incorporated by reference herein; and Liu, D. R. & Schultz, P.G. (1999) Progress toward the evolution of an organism with an expandedgenetic code. PNAS United States 96:4780-4785. Although uptake is easilyanalyzed with various assays, an alternative to designing unnaturalamino acids that are amenable to cellular uptake pathways is to providebiosynthetic pathways to create amino acids in vivo.

(i) Biosynthesis of Unnatural Amino Acids

Many biosynthetic pathways already exist in cells for the production ofamino acids and other compounds. While a biosynthetic method for aparticular unnatural amino acid may not exist in nature, including butnot limited to, in a cell, the invention provides such methods. Forexample, biosynthetic pathways for unnatural amino acids are optionallygenerated in host cell by adding new enzymes or modifying existing hostcell pathways. Additional new enzymes are optionally naturally occurringenzymes or artificially evolved enzymes. For example, the biosynthesisof p-aminophenylalanine (as presented in an example in WO 2002/085923entitled “In vivo incorporation of unnatural amino acids”) relies on theaddition of a combination of known enzymes from other organisms. Thegenes for these enzymes can be introduced into a eukaryotic cell bytransforming the cell with a plasmid comprising the genes. The genes,when expressed in the cell, provide an enzymatic pathway to synthesizethe desired compound. Examples of the types of enzymes that areoptionally added are provided in the examples below. Additional enzymessequences are found, for example, in Genbank. Artificially evolvedenzymes are also optionally added into a cell in the same manner. Inthis manner, the cellular machinery and resources of a cell aremanipulated to produce unnatural amino acids.

A variety of methods are available for producing novel enzymes for usein biosynthetic pathways or for evolution of existing pathways. Forexample, recursive recombination, including but not limited to, asdeveloped by Maxygen, Inc. (available on the World Wide Web atmaxygen.com), is optionally used to develop novel enzymes and pathways.See, e.g., Stemmer (1994), Rapid evolution of a protein in vitro by DNAshuffling, Nature 370(4):389-391; and, Stemmer, (1994), DNA shuffling byrandom fragmentation and reassembly: In vitro recombination formolecular evolution, Proc. Natl. Acad. Sci. USA., 91:10747-10751.Similarly DesignPath™, developed by Genencor (available on the WorldWide Web at genencor.com) is optionally used for metabolic pathwayengineering, including but not limited to, to engineer a pathway tocreate O-methyl-L-tyrosine in a cell. This technology reconstructsexisting pathways in host organisms using a combination of new genes,including but not limited to, those identified through functionalgenomics, and molecular evolution and design. Diversa Corporation(available on the World Wide Web at diversa.com) also providestechnology for rapidly screening libraries of genes and gene pathways,including but not limited to, to create new pathways.

Typically, the unnatural amino acid produced with an engineeredbiosynthetic pathway of the invention is produced in a concentrationsufficient for efficient protein biosynthesis, including but not limitedto, a natural cellular amount, but not to such a degree as to affect theconcentration of the other amino acids or exhaust cellular resources.Typical concentrations produced in vivo in this manner are about 10 mMto about 0.05 mM. Once a cell is transformed with a plasmid comprisingthe genes used to produce enzymes desired for a specific pathway and anunnatural amino acid is generated, in vivo selections are optionallyused to further optimize the production of the unnatural amino acid forboth ribosomal protein synthesis and cell growth.

(b) Polypeptides with Unnatural Amino Acids

The incorporation of an unnatural amino acid can be done for a varietyof purposes, including but not limited to, tailoring changes in proteinstructure and/or function, changing size, acidity, nucleophilicity,hydrogen bonding, hydrophobicity, accessibility of protease targetsites, targeting to a moiety (including but not limited to, for aprotein array), adding a biologically active molecule, attaching apolymer, attaching a radionuclide, modulating serum half-life,modulating tissue penetration (e.g. tumors), modulating activetransport, modulating tissue, cell or organ specificity or distribution,modulating immunogenicity, modulating protease resistance, etc. Proteinsthat include an unnatural amino acid can have enhanced or even entirelynew catalytic or biophysical properties. For example, the followingproperties are optionally modified by inclusion of an unnatural aminoacid into a protein: toxicity, biodistribution, structural properties,spectroscopic properties, chemical and/or photochemical properties,catalytic ability, half-life (including but not limited to, serumhalf-life), ability to react with other molecules, including but notlimited to, covalently or noncovalently, and the like. The compositionsincluding proteins that include at least one unnatural amino acid areuseful for, including but not limited to, novel therapeutics,diagnostics, catalytic enzymes, industrial enzymes, binding proteins(including but not limited to, antibodies), and including but notlimited to, the study of protein structure and function. See, e.g.,Dougherty, (2000) Unnatural Amino Acids as Probes of Protein Structureand Function, Current Opinion in Chemical Biology, 4:645-652.

In one aspect of the invention, a composition includes at least oneprotein with at least one, including but not limited to, at least two,at least three, at least four, at least five, at least six, at leastseven, at least eight, at least nine, or at least ten or more unnaturalamino acids. The unnatural amino acids can be the same or different,including but not limited to, there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 or more different sites in the protein that comprise 1, 2, 3, 4, 5,6, 7, 8, 9, or or more different unnatural amino acids. In anotheraspect, a composition includes a protein with at least one, but fewerthan all, of a particular amino acid present in the protein issubstituted with the unnatural amino acid. For a given protein with morethan one unnatural amino acids, the unnatural amino acids can beidentical or different (including but not limited to, the protein caninclude two or more different types of unnatural amino acids, or caninclude two of the same unnatural amino acid). For a given protein withmore than two unnatural amino acids, the unnatural amino acids can bethe same, different or a combination of a multiple unnatural amino acidof the same kind with at least one different unnatural amino acid.

Proteins or polypeptides of interest with at least one unnatural aminoacid are a feature of the invention. The invention also includespolypeptides or proteins with at least one unnatural amino acid producedusing the compositions and methods of the invention. An excipient(including but not limited to, a pharmaceutically acceptable excipient)can also be present with the protein.

By producing proteins or polypeptides of interest with at least oneunnatural amino acid in eukaryotic cells, proteins or polypeptides willtypically include eukaryotic post-translational modifications. Incertain embodiments, a protein includes at least one unnatural aminoacid and at least one post-translational modification that is made invivo by a eukaryotic cell, where the post-translational modification isnot made by a prokaryotic cell. For example, the post-translationmodification includes, including but not limited to, glycosylation,acetylation, acylation, lipid-modification, palmitoylation, palmitateaddition, phosphorylation, glycolipid-linkage modification,glycosylation, and the like. In one aspect, the post-translationalmodification includes attachment of an oligosaccharide (including butnot limited to, (GlcNAc-Man)₂-Man-GlcNAc-GlcNAc)) to an asparagine by aGlcNAc-asparagine linkage. See Table 1 which lists some examples ofN-linked oligosaccharides of eukaryotic proteins (additional residuescan also be present, which are not shown). In another aspect, thepost-translational modification includes attachment of anoligosaccharide (including but not limited to, Gal-GalNAc, Gal-GlcNAc,etc.) to a serine or threonine by a GalNAc-serine or GalNAc-threoninelinkage, or a GlcNAc-serine or a GlcNAc-threonine linkage. TABLE 1Examples of oligosaccharides through GlcNAc-linkage Type Base Structure2. HIGH- MANNOSE

3. HYBRID

4. COMPLEX

5. XYLOSE

In yet another aspect, the post-translation modification includesproteolytic processing of precursors (including but not limited to,calcitonin precursor, calcitonin gene-related peptide precursor,preproparathyroid hormone, preproinsulin, proinsulin,prepro-opiomelanocortin, pro-opiomelanocortin and the like), assemblyinto a multisubunit protein or macromolecular assembly, translation toanother site in the cell (including but not limited to, to organelles,such as the endoplasmic reticulum, the Golgi apparatus, the nucleus,lysosomes, peroxisomes, mitochondria, chloroplasts, vacuoles, etc., orthrough the secretory pathway). In certain embodiments, the proteincomprises a secretion or localization sequence, an epitope tag, a FLAGtag, a polyhistidine tag, a GST fusion, or the like. U.S. Pat. Nos.4,963,495 and 6,436,674, which are incorporated herein by reference,detail constructs designed to improve secretion of GH, e.g., hGHpolypeptides.

One advantage of an unnatural amino acid is that it presents additionalchemical moieties that can be used to add additional molecules. Thesemodifications can be made in vivo in a eukaryotic or non-eukaryoticcell, or in vitro. Thus, in certain embodiments, the post-translationalmodification is through the unnatural amino acid. For example, thepost-translational modification can be through anucleophilic-electrophilic reaction. Most reactions currently used forthe selective modification of proteins involve covalent bond formationbetween nucleophilic and electrophilic reaction partners, including butnot limited to the reaction of α-haloketones with histidine or cysteineside chains. Selectivity in these cases is determined by the number andaccessibility of the nucleophilic residues in the protein. In proteinsof the invention, other more selective reactions can be used such as thereaction of an unnatural keto-amino acid with hydrazides or aminooxycompounds, in vitro and in vivo. See, e.g., Cornish, et al., (1996) J.Am. Chem. Soc., 118:8150-8151; Mahal, et al., (1997) Science,276:1125-1128; Wang, et al., (2001) Science 292:498-500; Chin, et al.,(2002) J. Am. Chem. Soc. 124:9026-9027; Chin, et al., (2002) Proc. Natl.Acad. Sci., 99:11020-11024; Wang, et al., (2003) Proc. Natl. Acad. Sci.,100:56-61; Zhang, et al., (2003) Biochemistry, 42:6735-6746; and, Chin,et al., (2003) Science, 301:964-7, all of which are incorporated byreference herein. This allows the selective labeling of virtually anyprotein with a host of reagents including fluorophores, crosslinkingagents, saccharide derivatives and cytotoxic molecules. See also, U.S.Pat. No. 6,927,042 entitled “Glycoprotein synthesis,” which isincorporated by reference herein. Post-translational modifications,including but not limited to, through an azido amino acid, can also madethrough the Staudinger ligation (including but not limited to, withtriarylphosphine reagents). See, e.g., Kiick et al., (2002)Incorporation of azides into recombinant proteins for chemoselectivemodification by the Staudinger ligation, PNAS 99:19-24.

This invention provides another highly efficient method for theselective modification of proteins, which involves the geneticincorporation of unnatural amino acids, including but not limited to,containing an azide or alkynyl moiety into proteins in response to aselector codon. These amino acid side chains can then be modified by,including but not limited to, a Huisgen [3+2]cycloaddition reaction(see, e.g., Padwa, A. in Comprehensive Organic Synthesis, Vol. 4, (1991)Ed. Trost, B. M., Pergamon, Oxford, p. 1069-1109; and, Huisgen, R. in1,3-Dipolar Cycloaddition Chemistry, (1984) Ed. Padwa, A., Wiley, NewYork, p. 1-176) with, including but not limited to, alkynyl or azidederivatives, respectively. Because this method involves a cycloadditionrather than a nucleophilic substitution, proteins can be modified withextremely high selectivity. This reaction can be carried out at roomtemperature in aqueous conditions with excellent regioselectivity(1,4>1,5) by the addition of catalytic amounts of Cu(I) salts to thereaction mixture. See, e.g., Tornoe, et al., (2002) J. Org. Chem.67:3057-3064; and, Rostovtsev, et al., (2002) Angew. Chem. Int. Ed.41:2596-2599. Another method that can be used is the ligand exchange ona bisarsenic compound with a tetracysteine motif, see, e.g., Griffin, etal., (1998) Science 281:269-272.

A molecule that can be added to a protein of the invention through a[3+2]cycloaddition includes virtually any molecule with an azide oralkynyl derivative. Molecules include, but are not limited to, dyes,fluorophores, crosslinking agents, saccharide derivatives, polymers(including but not limited to, derivatives of polyethylene glycol),photocrosslinkers, cytotoxic compounds, affinity labels, derivatives ofbiotin, resins, beads, a second protein or polypeptide (or more),polynucleotide(s) (including but not limited to, DNA, RNA, etc.), metalchelators, cofactors, fatty acids, carbohydrates, and the like. Thesemolecules can be added to an unnatural amino acid with an alkynyl group,including but not limited to, p-propargyloxyphenylalanine, or azidogroup, including but not limited to, p-azido-phenylalanine,respectively.

V. In Vivo Generation of GH, e.g., hGH Polypeptides ComprisingNon-Genetically-Encoded Amino Acids

The GH, e.g., hGH polypeptides of the invention can be generated in vivousing modified tRNA and tRNA synthetases to add to or substitute aminoacids that are not encoded in naturally-occurring systems.

Methods for generating tRNAs and tRNA synthetases which use amino acidsthat are not encoded in naturally-occurring systems are described in,e.g., U.S. Patent Application Publications 2003/0082575 (Ser. No.10/126,927) and 2003/0108885 (Ser. No. 10/126,931) which areincorporated by reference herein. These methods involve generating atranslational machinery that functions independently of the synthetasesand tRNAs endogenous to the translation system (and are thereforesometimes referred to as “orthogonal”). Typically, the translationsystem comprises an orthogonal tRNA (O-tRNA) and an orthogonal aminoacyltRNA synthetase (O—RS). Typically, the O—RS preferentially aminoacylatesthe O-tRNA with at least one non-naturally occurring amino acid in thetranslation system and the O-tRNA recognizes at least one selector codonthat is not recognized by other tRNAs in the system. The translationsystem thus inserts the non-naturally-encoded amino acid into a proteinproduced in the system, in response to an encoded selector codon,thereby “substituting” an amino acid into a position in the encodedpolypeptide.

A wide variety of orthogonal tRNAs and aminoacyl tRNA synthetases havebeen described in the art for inserting particular synthetic amino acidsinto polypeptides, and are generally suitable for use in the presentinvention. For example, keto-specific O-tRNA/aminoacyl-tRNA synthetasesare described in Wang, L., et al., Proc. Natl. Acad. Sci. USA 100:56-61(2003) and Zhang, Z. et al., Biochem. 42(22):6735-6746 (2003). ExemplaryO—RS, or portions thereof, are encoded by polynucleotide sequences andinclude amino acid sequences disclosed in U.S. Patent ApplicationPublications 2003/0082575 and 2003/0108885, each incorporated herein byreference. Corresponding O-tRNA molecules for use with the O—RSs arealso described in U.S. Patent Application Publications 2003/0082575(Ser. No. 10/126,927) and 2003/0108885 (Ser. No. 10/126,931) which areincorporated by reference herein.

An example of an azide-specific O-tRNA/aminoacyl-tRNA synthetase systemis described in Chin, J. W., et al., J. Am. Chem. Soc. 124:9026-9027(2002). Exemplary O—RS sequences for p-azido-L-Phe include, but are notlimited to, nucleotide sequences SEQ ID NOs: 14-16 and 29-32 and aminoacid sequences SEQ ID NOs: 46-48 and 61-64 as disclosed in U.S. PatentApplication Publication 2003/0108885 (Ser. No. 10/126,931) which isincorporated by reference herein. Exemplary O-tRNA sequences suitablefor use in the present invention include, but are not limited to,nucleotide sequences SEQ ID NOs: 1-3 as disclosed in U.S. PatentApplication Publication 2003/0108885 (Ser. No. 10/126,931) which isincorporated by reference herein. Other examples ofO-tRNA/aminoacyl-tRNA synthetase pairs specific to particularnon-naturally encoded amino acids are described in U.S. PatentApplication Publication 2003/0082575 (Ser. No. 10/126,927) which isincorporated by reference herein. O—RS and O-tRNA that incorporate bothketo- and azide-containing amino acids in S. cerevisiae are described inChin, J. W., et al., Science 301:964-967 (2003).

Several other orthogonal pairs have been reported. Glutaminyl (see,e.g., Liu, D. R., and Schultz, P. G. (1999) Proc. Natl. Acad. Sci.U.S.A. 96:4780-4785), aspartyl (see, e.g., Pastrnak, M., et al., (2000)Helv. Chim. Acta 83:2277-2286), and tyrosyl (see, e.g., Ohno, S., etal., (1998) J. Biochem. (Tokyo, Jpn.) 124:1065-1068; and, Kowal, A. K.,et al., (2001) Proc. Natl. Acad. Sci. U.S.A. 98:2268-2273) systemsderived from S. cerevisiae tRNA's and synthetases have been describedfor the potential incorporation of unnatural amino acids in E. coli.Systems derived from the E. coli glutaminyl (see, e.g., Kowal, A. K., etal., (2001) Proc. Natl. Acad. Sci. U.S. A. 98:2268-2273) and tyrosyl(see, e.g., Edwards, H., and Schimmel, P. (1990) Mol. Cell. Biol.10:1633-1641) synthetases have been described for use in S. cerevisiae.The E. coli tyrosyl system has been used for the incorporation of3-iodo-L-tyrosine in vivo, in mammalian cells. See, Sakamoto, K., etal., (2002) Nucleic Acids Res. 30:4692-4699.

Use of O-tRNA/aminoacyl-tRNA synthetases involves selection of aspecific codon which encodes the non-naturally encoded amino acid. Whileany codon can be used, it is generally desirable to select a codon thatis rarely or never used in the cell in which the O-tRNA/aminoacyl-tRNAsynthetase is expressed. For example, exemplary codons include nonsensecodon such as stop codons (amber, ochre, and opal), four or more basecodons and other natural three-base codons that are rarely or unused.

Specific selector codon(s) can be introduced into appropriate positionsin the GH, e.g., hGH polynucleotide coding sequence using mutagenesismethods known in the art (including but not limited to, site-specificmutagenesis, cassette mutagenesis, restriction selection mutagenesis,etc.).

Methods for generating components of the protein biosynthetic machinery,such as O—RSs, O-tRNAs, and orthogonal O-tRNA/O—RS pairs that can beused to incorporate a non-naturally encoded amino acid are described inWang, L., et al., Science 292: 498-500 (2001); Chin, J. W., et al., J.Am. Chem. Soc. 124:9026-9027 (2002); Zhang, Z. et al., Biochemistry 42:6735-6746 (2003). Methods and compositions for the in vivo incorporationof non-naturally encoded amino acids are described in U.S. PatentApplication Publication 2003/0082575 (Ser. No. 10/126,927) which isincorporated by reference herein. Methods for selecting an orthogonaltRNA-tRNA synthetase pair for use in in vivo translation system of anorganism are also described in U.S. Patent Application Publications2003/0082575 (Ser. No. 10/126,927) and 2003/0108885 (Ser. No.10/126,931) which are incorporated by reference herein. PCT PublicationNo. WO 04/035743 entitled “Site Specific Incorporation of Keto AminoAcids into Proteins,” which is incorporated by reference herein in itsentirety, describes orthogonal RS and tRNA pairs for the incorporationof keto amino acids. PCT Publication No. WO 04/094593 entitled“Expanding the Eukaryotic Genetic Code,” which is incorporated byreference herein in its entirety, describes orthogonal RS and tRNA pairsfor the incorporation of non-naturally encoded amino acids in eukaryotichost cells.

Methods for producing at least one recombinant orthogonal aminoacyl-tRNAsynthetase (O—RS) comprise: (a) generating a library of (optionallymutant) RSs derived from at least one aminoacyl-tRNA synthetase (RS)from a first organism, including but not limited to, a prokaryoticorganism, such as Methanococcus jannaschii, Methanobacteriumthermoautotrophicum, Halobacterium, Escherichia coli, A. fulgidus, P.furiosus, P. horikoshii, A. pernix, T. thermophilus, or the like, or aeukaryotic organism; (b) selecting (and/or screening) the library of RSs(optionally mutant RSs) for members that aminoacylate an orthogonal tRNA(O-tRNA) in the presence of a non-naturally encoded amino acid and anatural amino acid, thereby providing a pool of active (optionallymutant) RSs; and/or, (c) selecting (optionally through negativeselection) the pool for active RSs (including but not limited to, mutantRSs) that preferentially aminoacylate the O-tRNA in the absence of thenon-naturally encoded amino acid, thereby providing the at least onerecombinant O—RS; wherein the at least one recombinant O—RSpreferentially aminoacylates the O-tRNA with the non-naturally encodedamino acid.

In one embodiment, the RS is an inactive RS. The inactive RS can begenerated by mutating an active RS. For example, the inactive RS can begenerated by mutating at least about 1, at least about 2, at least about3, at least about 4, at least about 5, at least about 6, or at leastabout 10 or more amino acids to different amino acids, including but notlimited to, alanine.

Libraries of mutant RSs can be generated using various techniques knownin the art, including but not limited to rational design based onprotein three dimensional RS structure, or mutagenesis of RS nucleotidesin a random or rational design technique. For example, the mutant RSscan be generated by site-specific mutations, random mutations, diversitygenerating recombination mutations, chimeric constructs, rational designand by other methods described herein or known in the art.

In one embodiment, selecting (and/or screening) the library of RSs(optionally mutant RSs) for members that are active, including but notlimited to, that aminoacylate an orthogonal tRNA (O-tRNA) in thepresence of a non-naturally encoded amino acid and a natural amino acid,includes: introducing a positive selection or screening marker,including but not limited to, an antibiotic resistance gene, or thelike, and the library of (optionally mutant) RSs into a plurality ofcells, wherein the positive selection and/or screening marker comprisesat least one selector codon, including but not limited to, an amber,ochre, or opal codon; growing the plurality of cells in the presence ofa selection agent; identifying cells that survive (or show a specificresponse) in the presence of the selection and/or screening agent bysuppressing the at least one selector codon in the positive selection orscreening marker, thereby providing a subset of positively selectedcells that contains the pool of active (optionally mutant) RSs.Optionally, the selection and/or screening agent concentration can bevaried.

In one aspect, the positive selection marker is a chloramphenicolacetyltransferase (CAT) gene and the selector codon is an amber stopcodon in the CAT gene. Optionally, the positive selection marker is aβ-lactamase gene and the selector codon is an amber stop codon in theβ-lactamase gene. In another aspect the positive screening markercomprises a fluorescent or luminescent screening marker or an affinitybased screening marker (including but not limited to, a cell surfacemarker).

In one embodiment, negatively selecting or screening the pool for activeRSs (optionally mutants) that preferentially aminoacylate the O-tRNA inthe absence of the non-naturally encoded amino acid includes:introducing a negative selection or screening marker with the pool ofactive (optionally mutant) RSs from the positive selection or screeninginto a plurality of cells of a second organism, wherein the negativeselection or screening marker comprises at least one selector codon(including but not limited to, an antibiotic resistance gene, includingbut not limited to, a chloramphenicol acetyltransferase (CAT) gene);and, identifying cells that survive or show a specific screeningresponse in a first medium supplemented with the non-naturally encodedamino acid and a screening or selection agent, but fail to survive or toshow the specific response in a second medium not supplemented with thenon-naturally encoded amino acid and the selection or screening agent,thereby providing surviving cells or screened cells with the at leastone recombinant O—RS. For example, a CAT identification protocoloptionally acts as a positive selection and/or a negative screening indetermination of appropriate O—RS recombinants. For instance, a pool ofclones is optionally replicated on growth plates containing CAT (whichcomprises at least one selector codon) either with or without one ormore non-naturally encoded amino acid. Colonies growing exclusively onthe plates containing non-naturally encoded amino acids are thusregarded as containing recombinant O—RS. In one aspect, theconcentration of the selection (and/or screening) agent is varied. Insome aspects the first and second organisms are different. Thus, thefirst and/or second organism optionally comprises: a prokaryote, aeukaryote, a mammal, an Escherichia coli, a fungi, a yeast, anarchaebacterium, a eubacterium, a plant, an insect, a protist, etc. Inother embodiments, the screening marker comprises a fluorescent orluminescent screening marker or an affinity based screening marker.

In another embodiment, screening or selecting (including but not limitedto, negatively selecting) the pool for active (optionally mutant) RSsincludes: isolating the pool of active mutant RSs from the positiveselection step (b); introducing a negative selection or screeningmarker, wherein the negative selection or screening marker comprises atleast one selector codon (including but not limited to, a toxic markergene, including but not limited to, a ribonuclease barnase gene,comprising at least one selector codon), and the pool of active(optionally mutant) RSs into a plurality of cells of a second organism;and identifying cells that survive or show a specific screening responsein a first medium not supplemented with the non-naturally encoded aminoacid, but fail to survive or show a specific screening response in asecond medium supplemented with the non-naturally encoded amino acid,thereby providing surviving or screened cells with the at least onerecombinant O—RS, wherein the at least one recombinant O—RS is specificfor the non-naturally encoded amino acid. In one aspect, the at leastone selector codon comprises about two or more selector codons. Suchembodiments optionally can include wherein the at least one selectorcodon comprises two or more selector codons, and wherein the first andsecond organism are different (including but not limited to, eachorganism is optionally, including but not limited to, a prokaryote, aeukaryote, a mammal, an Escherichia coli, a fungi, a yeast, anarchaebacteria, a eubacteria, a plant, an insect, a protist, etc.).Also, some aspects include wherein the negative selection markercomprises a ribonuclease barnase gene (which comprises at least oneselector codon). Other aspects include wherein the screening markeroptionally comprises a fluorescent or luminescent screening marker or anaffinity based screening marker. In the embodiments herein, thescreenings and/or selections optionally include variation of thescreening and/or selection stringency.

In one embodiment, the methods for producing at least one recombinantorthogonal aminoacyl-tRNA synthetase (O—RS) can further comprise: (d)isolating the at least one recombinant O—RS; (e) generating a second setof O—RS (optionally mutated) derived from the at least one recombinantO—RS; and, (f) repeating steps (b) and (c) until a mutated O—RS isobtained that comprises an ability to preferentially aminoacylate theO-tRNA. Optionally, steps (d)-(f) are repeated, including but notlimited to, at least about two times. In one aspect, the second set ofmutated O—RS derived from at least one recombinant O—RS can be generatedby mutagenesis, including but not limited to, random mutagenesis,site-specific mutagenesis, recombination or a combination thereof.

The stringency of the selection/screening steps, including but notlimited to, the positive selection/screening step (b), the negativeselection/screening step (c) or both the positive and negativeselection/screening steps (b) and (c), in the above-described methods,optionally includes varying the selection/screening stringency. Inanother embodiment, the positive selection/screening step (b), thenegative selection/screening step (c) or both the positive and negativeselection/screening steps (b) and (c) comprise using a reporter, whereinthe reporter is detected by fluorescence-activated cell sorting (FACS)or wherein the reporter is detected by luminescence. Optionally, thereporter is displayed on a cell surface, on a phage display or the likeand selected based upon affinity or catalytic activity involving thenon-naturally encoded amino acid or an analogue. In one embodiment, themutated synthetase is displayed on a cell surface, on a phage display orthe like.

Methods for producing a recombinant orthogonal tRNA (O-tRNA) include:(a) generating a library of mutant tRNAs derived from at least one tRNA,including but not limited to, a suppressor tRNA, from a first organism;(b) selecting (including but not limited to, negatively selecting) orscreening the library for (optionally mutant) tRNAs that areaminoacylated by an aminoacyl-tRNA synthetase (RS) from a secondorganism in the absence of a RS from the first organism, therebyproviding a pool of tRNAs (optionally mutant); and, (c) selecting orscreening the pool of tRNAs (optionally mutant) for members that areaminoacylated by an introduced orthogonal RS(O—RS), thereby providing atleast one recombinant O-tRNA; wherein the at least one recombinantO-tRNA recognizes a selector codon and is not efficiency recognized bythe RS from the second organism and is preferentially aminoacylated bythe O—RS. In some embodiments the at least one tRNA is a suppressor tRNAand/or comprises a unique three base codon of natural and/or unnaturalbases, or is a nonsense codon, a rare codon, an unnatural codon, a codoncomprising at least 4 bases, an amber codon, an ochre codon, or an opalstop codon. In one embodiment, the recombinant O-tRNA possesses animprovement of orthogonality. It will be appreciated that in someembodiments, O-tRNA is optionally imported into a first organism from asecond organism without the need for modification. In variousembodiments, the first and second organisms are either the same ordifferent and are optionally chosen from, including but not limited to,prokaryotes (including but not limited to, Methanococcus jannaschii,Methanobacterium thermoautotrophicum, Escherichia coli, Halobacterium,etc.), eukaryotes, mammals, fungi, yeasts, archaebacteria, eubacteria,plants, insects, protists, etc. Additionally, the recombinant tRNA isoptionally aminoacylated by a non-naturally encoded amino acid, whereinthe non-naturally encoded amino acid is biosynthesized in vivo eithernaturally or through genetic manipulation. The non-naturally encodedamino acid is optionally added to a growth medium for at least the firstor second organism.

In one aspect, selecting (including but not limited to, negativelyselecting) or screening the library for (optionally mutant) tRNAs thatare aminoacylated by an aminoacyl-tRNA synthetase (step (b)) includes:introducing a toxic marker gene, wherein the toxic marker gene comprisesat least one of the selector codons (or a gene that leads to theproduction of a toxic or static agent or a gene essential to theorganism wherein such marker gene comprises at least one selector codon)and the library of (optionally mutant) tRNAs into a plurality of cellsfrom the second organism; and, selecting surviving cells, wherein thesurviving cells contain the pool of (optionally mutant) tRNAs comprisingat least one orthogonal tRNA or nonfunctional tRNA. For example,surviving cells can be selected by using a comparison ratio cell densityassay.

In another aspect, the toxic marker gene can include two or moreselector codons. In another embodiment of the methods, the toxic markergene is a ribonuclease barnase gene, where the ribonuclease barnase genecomprises at least one amber codon. Optionally, the ribonuclease barnasegene can include two or more amber codons.

In one embodiment, selecting or screening the pool of (optionallymutant) tRNAs for members that are aminoacylated by an introducedorthogonal RS(O—RS) can include: introducing a positive selection orscreening marker gene, wherein the positive marker gene comprises a drugresistance gene (including but not limited to, β-lactamase gene,comprising at least one of the selector codons, such as at least oneamber stop codon) or a gene essential to the organism, or a gene thatleads to detoxification of a toxic agent, along with the O—RS, and thepool of (optionally mutant) tRNAs into a plurality of cells from thesecond organism; and, identifying surviving or screened cells grown inthe presence of a selection or screening agent, including but notlimited to, an antibiotic, thereby providing a pool of cells possessingthe at least one recombinant tRNA, where the at least one recombinanttRNA is aminoacylated by the O—RS and inserts an amino acid into atranslation product encoded by the positive marker gene, in response tothe at least one selector codons. In another embodiment, theconcentration of the selection and/or screening agent is varied.

Methods for generating specific O-tRNA/O—RS pairs are provided. Methodsinclude: (a) generating a library of mutant tRNAs derived from at leastone tRNA from a first organism; (b) negatively selecting or screeningthe library for (optionally mutant) tRNAs that are aminoacylated by anaminoacyl-tRNA synthetase (RS) from a second organism in the absence ofa RS from the first organism, thereby providing a pool of (optionallymutant) tRNAs; (c) selecting or screening the pool of (optionallymutant) tRNAs for members that are aminoacylated by an introducedorthogonal RS(O—RS), thereby providing at least one recombinant O-tRNA.The at least one recombinant O-tRNA recognizes a selector codon and isnot efficiency recognized by the RS from the second organism and ispreferentially aminoacylated by the O—RS. The method also includes (d)generating a library of (optionally mutant) RSs derived from at leastone aminoacyl-tRNA synthetase (RS) from a third organism; (e) selectingor screening the library of mutant RSs for members that preferentiallyaminoacylate the at least one recombinant O-tRNA in the presence of anon-naturally encoded amino acid and a natural amino acid, therebyproviding a pool of active (optionally mutant) RSs; and, (f) negativelyselecting or screening the pool for active (optionally mutant) RSs thatpreferentially aminoacylate the at least one recombinant O-tRNA in theabsence of the non-naturally encoded amino acid, thereby providing theat least one specific O-tRNA/O—RS pair, wherein the at least onespecific O-tRNA/O—RS pair comprises at least one recombinant O—RS thatis specific for the non-naturally encoded amino acid and the at leastone recombinant O-tRNA. Specific O-tRNA/O—RS pairs produced by themethods are included. For example, the specific O-tRNA/O—RS pair caninclude, including but not limited to, a mutRNATyr-mutTyrRS pair, suchas a mutRNATyr-SS12TyrRS pair, a mutRNALeu-mutLeuRS pair, amutRNAThr-mutThrRS pair, a mutRNAGlu-mutGluRS pair, or the like.Additionally, such methods include wherein the first and third organismare the same (including but not limited to, Methanococcus jannaschii).

Methods for selecting an orthogonal tRNA-tRNA synthetase pair for use inan in vivo translation system of a second organism are also included inthe present invention. The methods include: introducing a marker gene, atRNA and an aminoacyl-tRNA synthetase (RS) isolated or derived from afirst organism into a first set of cells from the second organism;introducing the marker gene and the tRNA into a duplicate cell set froma second organism; and, selecting for surviving cells in the first setthat fail to survive in the duplicate cell set or screening for cellsshowing a specific screening response that fail to give such response inthe duplicate cell set, wherein the first set and the duplicate cell setare grown in the presence of a selection or screening agent, wherein thesurviving or screened cells comprise the orthogonal tRNA-tRNA synthetasepair for use in the in the in vivo translation system of the secondorganism. In one embodiment, comparing and selecting or screeningincludes an in vivo complementation assay. The concentration of theselection or screening agent can be varied.

The organisms of the present invention comprise a variety of organismand a variety of combinations. For example, the first and the secondorganisms of the methods of the present invention can be the same ordifferent. In one embodiment, the organisms are optionally a prokaryoticorganism, including but not limited to, Methanococcus jannaschii,Methanobacterium thermoautotrophicum, Halobacterium, Escherichia coli,A. fulgidus, P. furiosus, P. horikoshii, A. pernix, T. thermophilus, orthe like. Alternatively, the organisms optionally comprise a eukaryoticorganism, including but not limited to, plants (including but notlimited to, complex plants such as monocots, or dicots), algae,protists, fungi (including but not limited to, yeast, etc), animals(including but not limited to, mammals, insects, arthropods, etc.), orthe like. In another embodiment, the second organism is a prokaryoticorganism, including but not limited to, Methanococcus jannaschii,Methanobacterium thermoautotrophicum, Halobacterium, Escherichia coli,A. fulgidus, Halobacterium, P. furiosus, P. horikoshii, A. pernix, T.thermophilus, or the like. Alternatively, the second organism can be aeukaryotic organism, including but not limited to, a yeast, a animalcell, a plant cell, a fungus, a mammalian cell, or the like. In variousembodiments the first and second organisms are different.

VI. Location of Non-Naturally-Occurring Amino Acids in GH, e.g., hGHpolypeptides

The present invention contemplates incorporation of one or morenon-naturally-occurring amino acids into GH, e.g., hGH polypeptides. Oneor more non-naturally-occurring amino acids may be incorporated at aparticular position which does not disrupt activity of the polypeptide.This can be achieved by making “conservative” substitutions, includingbut not limited to, substituting hydrophobic amino acids withhydrophobic amino acids, bulky amino acids for bulky amino acids,hydrophilic amino acids for hydrophilic amino acids and/or inserting thenon-naturally-occurring amino acid in a location that is not requiredfor activity.

Regions of GH, e.g., hGH can be illustrated as follows, wherein theamino acid positions in hGH are indicated in the middle row (SEQ ID NO:2):

A variety of biochemical and structural approaches can be employed toselect the desired sites for substitution with a non-naturally encodedamino acid within the GH, e.g., hGH polypeptide. It is readily apparentto those of ordinary skill in the art that any position of thepolypeptide chain is suitable for selection to incorporate anon-naturally encoded amino acid, and selection may be based on rationaldesign or by random selection for any or no particular desired purpose.Selection of desired sites may be for producing a GH, e.g., hGH moleculehaving any desired property or activity, including but not limited to,agonists, super-agonists, inverse agonists, antagonists, receptorbinding modulators, receptor activity modulators, dimer or multimerformation, no change to activity or property compared to the nativemolecule, or manipulating any physical or chemical property of thepolypeptide such as solubility, aggregation, or stability. For example,locations in the polypeptide required for biological activity of GH,e.g., hGH polypeptides can be identified using point mutation analysis,alanine scanning or homolog scanning methods known in the art. See,e.g., Cunningham, B. and Wells, J., Science, 244:1081-1085 (1989)(identifying 14 residues that are critical for GH, e.g., hGHbioactivity) and Cunningham, B., et al. Science 243: 1330-1336 (1989)(identifying antibody and receptor epitopes using homolog scanningmutagenesis). U.S. Pat. Nos. 5,580,723; 5,834,250; 6,013,478; 6,428,954;and 6,451,561, which are incorporated by reference herein, describemethods for the systematic analysis of the structure and function ofpolypeptides such as hGH by identifying active domains which influencethe activity of the polypeptide with a target substance. Residues otherthan those identified as critical to biological activity by alanine orhomolog scanning mutagenesis may be good candidates for substitutionwith a non-naturally encoded amino acid depending on the desiredactivity sought for the polypeptide. Alternatively, the sites identifiedas critical to biological activity may also be good candidates forsubstitution with a non-naturally encoded amino acid, again depending onthe desired activity sought for the polypeptide. Another alternativewould be to simply make serial substitutions in each position on thepolypeptide chain with a non-naturally encoded amino acid and observethe effect on the activities of the polypeptide. It is readily apparentto those of ordinary skill in the art that any means, technique, ormethod for selecting a position for substitution with a non-naturalamino acid into any polypeptide is suitable for use in the presentinvention.

The structure and activity of naturally-occurring mutants of hGHpolypeptides that contain deletions can also be examined to determineregions of the protein that are likely to be tolerant of substitutionwith a non-naturally encoded amino acid. See, e.g., Kostyo et al.,Biochem. Biophys. Acta, 925: 314 (1987); Lewis, U., et al., J. Biol.Chem., 253:2679-2687 (1978) for hGH. In a similar manner, proteasedigestion and monoclonal antibodies can be used to identify regions ofhGH that are responsible for binding the hGH receptor. See, e.g.,Cunningham, B., et al. Science 243: 1330-1336 (1989); Mills, J., et al.,Endocrinology, 107:391-399 (1980); Li, C., Mol. Cell. Biochem., 46:31-41(1982) (indicating that amino acids between residues 134-149 can bedeleted without a loss of activity). Once residues that are likely to beintolerant to substitution with non-naturally encoded amino acids havebeen eliminated, the impact of proposed substitutions at each of theremaining positions can be examined from the three-dimensional crystalstructure of the hGH and its binding proteins. See de Vos, A., et al.,Science, 255:306-3.12 (1992) for hGH; all crystal structures of hGH areavailable in the Protein Data Bank (including 3HHR, 1AXI, and 1HWG)(PDB, available on the World Wide Web at rcsb.org), a centralizeddatabase containing three-dimensional structural data of large moleculesof proteins and nucleic acids. Models may be made investigating thesecondary and tertiary structure of polypeptides, if three-dimensionalstructural data is not available. Thus, those of ordinary skill in theart can readily identify amino acid positions that can be substitutedwith non-naturally encoded amino acids.

In some embodiments, the GH, e.g., hGH polypeptides of the inventioncomprise one or more non-naturally occurring amino acids positioned in aregion of the protein that does not disrupt the helices or beta sheetsecondary structure of the polypeptide.

Exemplary residues of incorporation of a non-naturally encoded aminoacid may be those that are excluded from potential receptor bindingregions (including but not limited to, Site I and Site II), may be fullyor partially solvent exposed, have minimal or no hydrogen-bondinginteractions with nearby residues, may be minimally exposed to nearbyreactive residues, and may be in regions that are highly flexible(including but not limited to, C-D loop) or structurally rigid(including but not limited to, B helix) as predicted by thethree-dimensional, crystal structure, secondary, tertiary, or quaternarystructure of hGH, bound or unbound to its receptor.

In some embodiments, one or more non-naturally encoded amino acids areincorporated at any position in one or more of the following regionscorresponding to secondary structures in hGH as follows: positionscorresponding to 1-5 (N-terminus), 6-33 (A helix), 34-74 (region betweenA helix and B helix, the A-B loop), 75-96 (B helix), 97-105 (regionbetween B helix and C helix, the B-C loop), 106-129 (C helix), 130-153(region between C helix and D helix, the C-D loop), 1.54-183 (D helix),184-191 (C-terminus) from SEQ ID NO: 2. In other embodiments, GHpolypeptides, e.g., hGH polypeptides of the invention comprise at leastone non-naturally-occurring amino acid substituted for at least oneamino acid located in at least one region of GH, e.g., hGH selected fromthe group consisting regions corresponding to the N-terminus (1-5), theN-terminal end of the A-B loop (32-46); the B-C loop (97-105), the C-Dloop (132-149), and the C-terminus (184-191) of SEQ ID NO: 2. In someembodiments, one or more non-naturally encoded amino acids areincorporated at one or more of the following positions of GH, e.g., hGHcorresponding to: before position 1 (i.e. at the N-terminus), 1, 2, 3,4, 5, 8, 9, 11, 12, 15, 16, 19, 22, 29, 30, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 52, 55, 57, 59, 65, 66, 69,70, 71, 74, 88, 91, 92, 94, 95, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 111, 112, 113, 115, 116, 119, 120, 122, 123,126, 127, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 158, 159, 161, 168, 172, 183, 184, 185, 186, 187, 188, 189,190, 191, 192 (i.e., at the carboxyl terminus of the protein) of SEQ IDNO: 2 or the corresponding amino acids of SEQ ID NO: 1 or 3.

Exemplary sites of incorporation of one or more non-naturally encodedamino acids include sites corresponding to 29, 30, 33, 34, 35, 37, 39,40, 49, 57, 59, 66, 69, 70, 71, 74, 88, 91, 92, 94, 95, 98, 99, 101,103, 107, 108, 111, 122, 126, 129, 130, 131, 133, 134, 135, 136, 137,139, 140, 141, 142, 143, 145, 147, 154, 155, 156, 159, 183, 186, and187, or any combination thereof from SEQ ID NO: 2 or the correspondingamino acids of SEQ ID NO: 1 or 3.

A subset of exemplary sites for incorporation of one or morenon-naturally encoded amino acid include sites corresponding to 29, 33,35, 37, 39, 49, 57, 69, 70, 71, 74, 88, 91, 92, 94, 95, 98, 99, 101,103, 107, 108, 111, 129, 130, 131, 133, 134, 135, 136, 137, 139, 140,141, 142, 143, 145, 147, 154, 155, 156, 186, and 187, or any combinationthereof from SEQ ID NO: 2 or the corresponding amino acids of SEQ ID NO:1 or 3. An examination of the crystal structure of GH, e.g., hGH and itsinteractions with the GH, e.g., hGH receptor indicates that the sidechains of these amino acid residues are fully or partially accessible tosolvent and the side chain of a non-naturally encoded amino acid maypoint away from the protein surface and out into the solvent.

Exemplary positions for incorporation of one or more non-naturallyencoded amino acids include sites corresponding to 35, 88, 91, 92, 94,95, 99, 101, 103, 111, 131, 133, 134, 135, 136, 139, 140, 143, 145, and155, or any combination thereof from SEQ ID NO: 2 or the correspondingamino acids of SEQ ID NO: 1 or 3. An examination of the crystalstructure of GH, e.g., hGH and its interactions with the GH, e.g., hGHreceptor indicates that the side chains of these amino acid residues arefully exposed to the solvent and the side chain of the native residuepoints out into the solvent.

A subset of exemplary sites for incorporation of one or morenon-naturally encoded amino acids include sites corresponding to 30, 74,103, or any combination thereof, from SEQ ID NO: 2 or the correspondingamino acids of SEQ ID NO: 1 or 3. Another subset of exemplary sites forincorporation of one or more non-naturally encoded amino acids includesites corresponding to 35, 92, 143, 145, or any combination thereof,from SEQ ID NO: 2 or the corresponding amino acids of SEQ ID NO: 1 or 3.A further subset of exemplary sites for incorporation of one or morenon-naturally encoded amino acids include sites corresponding to 35, 92,131, 134, 143, 145, or any combination thereof, from SEQ ID NO: 2 or thecorresponding amino acids of SEQ ID NO: 1 or 3. Still a further subsetof exemplary sites for incorporation of one or more non-naturallyencoded amino acids include sites corresponding to 30, 35, 74, 92, 103,145, or any combination thereof, from SEQ ID NO: 2 or the correspondingamino acids of SEQ ID NO: 1 or 3. Yet a further subset of exemplarysites for incorporation of one or more non-naturally encoded amino acidsinclude sites corresponding to 35, 92, 143, 145, or any combinationthereof, from SEQ ID NO: 2 or the corresponding amino acids of SEQ IDNO: 1 or 3. In certain embodiments, sites for incorporation of one ormore non-naturally encoded amino acids include a site corresponding to35 from SEQ ID NO: 2 or the corresponding amino acids of SEQ ID NO: 1 or3.

In some embodiments, at least one of the non-naturally encoded aminoacids incorporated into the GH, e.g., hGH, contains a carbonyl group,e.g., a ketone group. In certain embodiments, at least one of thenon-naturally encoded amino acids incorporated into the GH, e.g., hGH ispara-acetylphenylalanine. In some embodiments in which the GH, e.g., hGHcontains a plurality of non-naturally-encoded amino acids, more than oneof the non-naturally-encoded amino acids incorporated into the GH, e.g.,hGH is para-acetylphenylalanine. In some embodiments in which the GH,e.g., hGH contains a plurality of non-naturally-encoded amino acids,substantially all of the non-naturally-encoded amino acids incorporatedinto the GH, e.g., hGH are para-acetylphenylalanine.

In some embodiments, the non-naturally occurring amino acid is linked toa water soluble polymer at one or more positions, including but notlimited to, positions corresponding to: before position 1 (i.e. at theN-terminus), 1, 2, 3, 4, 5, 8, 9, 11, 12, 15, 16, 19, 22, 29, 30, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 52,55, 57, 59, 65, 66, 69, 70, 71, 74, 88, 91, 92, 94, 95, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 111, 112, 113, 115, 116,119, 120, 122, 123, 126, 127, 129, 130, 131, 132, 133, 134, 135, 136,137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 158, 159, 161, 168, 172, 183, 184, 185,186, 187, 188, 189, 190, 191, 192 (i.e., at the carboxyl terminus of theprotein) (SEQ ID NO: 2 or the corresponding amino acids of SEQ ID NO: 1or 3). In some embodiments, the non-naturally occurring amino acid islinked to a water soluble polymer at positions including but not limitedto, positions corresponding to one or more of these positions: 30, 35,74, 92, 103, 143, 145 (SEQ ID NO: 2 or the corresponding amino acids ofSEQ ID NO: 1 or 3). In some embodiments, the non-naturally occurringamino acid is linked to a water soluble polymer at positions includingbut not limited to, positions corresponding to one or more of thesepositions: 35, 92, 143, 145 (SEQ ID NO: 2 or the corresponding aminoacids of SEQ ID NO: 1 or 3). In some embodiments, the non-naturallyoccurring amino acid is linked to a water soluble polymer at positionsincluding but not limited to, positions corresponding to one or more ofthese positions: 35, 92, 131, 134, 143, 145, or any combination thereof,from SEQ ID NO: 2 or the corresponding amino acids of SEQ ID NO: 1 or 3.In some embodiments, the non-naturally occurring amino acid is linked toa water soluble polymer at positions including but not limited to,positions corresponding to one or more of these positions: 30, 35, 74,92, 103, 145, or any combination thereof, from SEQ ID NO: 2 or thecorresponding amino acids of SEQ ID NO: 1 or 3. In some embodiments, thenon-naturally occurring amino acid is linked to a water soluble polymerat positions including but not limited to, positions corresponding toone or more of these positions: 35, 92, 143, 145, or any combinationthereof, from SEQ ID NO: 2 or the corresponding amino acids of SEQ IDNO: 1 or 3. In some embodiments, the non-naturally occurring amino acidis linked to a water-soluble polymer at a position corresponding to, butnot limited to, position 35 from SEQ ID NO: 2 or the corresponding aminoacids of SEQ ID NO: 1 or 3 is linked to a water-soluble polymer.

In some embodiments the water-soluble polymer(s) linked to the GH, e.g.,hGH, include one or more polyethylene glycol molecules (PEGs). Thepolymer, e.g., PEG, may be linear or branched. Typically, linearpolymers, e.g., PEGs, used in the invention can have a MW of about 0.1to about 100 kDa, or about 1 to about 60 kDa, or about 20 to about 40kDa, or about 30 kDa. Typically, branched polymers, e.g., PEGs, used inthe invention can have a MW of about 1 to about 100 kDa, or about 30 toabout 50 kDa, or about 40 kDa. Polymers such as PEGs are describedfurther herein. In certain embodiments, the linkage between the GH,e.g., hGH and the water-soluble polymer, e.g., PEG, is an oxime bond.

Certain embodiments of the invention encompass compositions that includea GH, e.g., hGH, linked to at least one water-soluble polymer by acovalent bond, where the covalent bond is an oxime bond. In someembodiments, the water-soluble polymer is a PEG, e.g., a linear PEG. Insome embodiments encompassing at least one linear PEG linked by an oximebond to a GH, e.g., hGH, the PEG can have a MW of about 0.1 to about 100kDa, or about 1 to about 60 kDa, or about 20 to about 40 kDa, or about30 kDa. In certain embodiments encompassing a linear PEG linked by anoxime bond to a GH, e.g., hGH, the PEG has a MW of about 30 kDa. In someembodiments encompassing at least one branched PEG linked by an oximebond to a GH, e.g., hGH, the PEG can have a MW of about 1 to about 100kDa or about 30 to about 50 kDa, or about 40 kDa. In certain embodimentsencompassing a branched PEG linked by an oxime bond to a GH, e.g., hGH,the PEG has a MW of about 40 kDa. In some embodiments, the GH is a GH,e.g., hGH and in certain of these embodiments, the GH, e.g., hGH has asequence that is at least about 80% identical to SEQ ID NO: 2; in someembodiments the GH, e.g., hGH has a sequence that is the sequence of SEQID NO: 2. In some embodiments, the GH, e.g., hGH, contains at least onenon-naturally-encoded amino acid; in some of these embodiments, at leastone oxime bond is between the non-naturally-encoded amino acid and atleast one water-soluble polymer. In some embodiments, thenon-naturally-encoded amino acid contains a carbonyl group, such as aketone group; in some embodiments, the non-naturally-encoded amino acidis para-acetylphenylalanine. In some embodiments, thepara-acetylphenylalanine is substituted at a position corresponding toposition 35 of SEQ ID NO: 2.

Thus, in some embodiments, the invention provides a GH, e.g., hGH,linked to at least one water-soluble polymer, e.g., a PEG, by a covalentbond, where the covalent bond is an oxime bond. In certain embodiments,the water-soluble polymer is a PEG and the PEG is a linear PEG. In theseembodiments, the linear PEG has a MW of about 0.1 to about 100 kDa, orabout 1 to about 60 kDa, or about 20 to about 40 kDa, or about 30 kDa.In certain embodiments encompassing a linear PEG linked by an oxime bondto a GH, e.g., hGH, the PEG has a MW of about 30 kDa. In certainembodiments, the water-soluble polymer is a PEG that is a branched PEG.In these embodiments, the branched PEG has a MW of about 1 to about 100kDa, or about 30 to about 50 kDa, or about 40 kDa. In certainembodiments encompassing a branched PEG linked by an oxime bond to a GH,e.g., hGH, the PEG has a MW of about 40 kDa.

In some embodiments, the invention provides a GH, e.g., hGH, where theGH, e.g., hGH contains a non-naturally encoded amino acid, where the GHis linked to at least one water-soluble polymer, e.g., a PEG, by acovalent bond, and where the covalent bond is an oxime bond between thenon-naturally encoded amino acid and the water-soluble polymer, e.g.,PEG. In some embodiments, the non-naturally-encoded amino acid isincorporated into the GH, e.g., hGH, at a position corresponding toposition 35 of SEQ ID NO: 2. In certain embodiments where thewater-soluble polymer is a PEG, the PEG is a linear PEG. In theseembodiments, the linear PEG has a MW of about 0.1 to about 100 kDa, orabout 1 to about 60 kDa, or about 20 to about 40 kDa, or about 30 kDa.In certain embodiments encompassing a linear PEG linked by an oxime bondto a GH, e.g., hGH, the PEG has a MW of about 30 kDa. In certainembodiments where the water-soluble polymer is a PEG, the PEG is abranched PEG. In these embodiments, the branched PEG has a MW of about 1to about 100 kDa, or about 30 to about 50 kDa, or about 40 kDa. Incertain embodiments encompassing a branched PEG linked by an oxime bondto a GH, e.g., hGH, the PEG has a MW of about 40 kDa.

In some embodiments, the invention provides a GH, e.g., hGH, where theGH, e.g., hGH contains a non-naturally encoded amino acid that is acarbonyl-containing non-naturally encoded amino acid, where the GH islinked to at least one water-soluble polymer, e.g., a PEG, by a covalentbond, and where the covalent bond is an oxime bond between thenon-naturally encoded carbonyl-containing amino acid and thewater-soluble polymer, e.g., PEG. In some embodiments, thenon-naturally-encoded carbonyl-containing amino acid is incorporatedinto the GH, e.g., hGH, at a position corresponding to position 35 ofSEQ ID NO: 2. In certain embodiments where the water-soluble polymer isa PEG, the PEG is a linear PEG. In these embodiments, the linear PEG hasa MW of about 0.1 to about 100 kDa, or about 1 to about 60 kDa, or about20 to about 40 kDa, or about 30 kDa. In certain embodiments encompassinga linear PEG linked by an oxime bond to a GH, e.g., hGH, the PEG has aMW of about 30 kDa. In certain embodiments where the water-solublepolymer is a PEG, the PEG is a branched PEG. In these embodiments, thebranched PEG has a MW of about 1 to about 100 kDa, or about 30 to about50 kDa, or about 40 kDa. In certain embodiments encompassing a branchedPEG linked by an oxime bond to a GH, e.g., hGH, the PEG has a MW ofabout 40 kDa.

In some embodiments, the invention provides a GH, e.g., hGH, thatcontains a non-naturally encoded amino acid that includes a ketonegroup, where the GH is linked to at least one water-soluble polymer,e.g., a PEG, by a covalent bond, and where the covalent bond is an oximebond between the non-naturally encoded amino acid containing a ketonegroup and the water-soluble polymer, e.g., PEG. In some embodiments, thenon-naturally-encoded amino acid containing a ketone group isincorporated into the GH, e.g., hGH, at a position corresponding toposition 35 of SEQ ID NO: 2. In certain embodiments where thewater-soluble polymer is a PEG, the PEG is a linear PEG. In theseembodiments, the linear PEG has a MW of about 0.1 to about 100 kDa, orabout 1 to about 60 kDa, or about 20 to about 40 kDa, or about 30 kDa.In certain embodiments encompassing a linear PEG linked by an oxime bondto a GH, e.g., hGH, the PEG has a MW of about 30 kDa. In certainembodiments where the water-soluble polymer is a PEG, the PEG is abranched PEG. In these embodiments, the branched PEG has a MW of about 1to about 100 kDa, or about 30 to about 50 kDa, or about 40 kDa. Incertain embodiments encompassing a branched PEG linked by an oxime bondto a GH, e.g., hGH, the PEG has a MW of about 40 kDa.

In some embodiments, the invention provides a GH, e.g., hGH, thatcontains a non-naturally encoded amino acid that is apara-acetylphenylalanine, where the GH linked to at least onewater-soluble polymer, e.g., a PEG, by a covalent bond, and where thecovalent bond is an oxime bond between the para-acetylphenylalanine andthe water-soluble polymer, e.g., PEG. In some embodiments, thepara-acetylphenylalanine is incorporated into the GH, e.g., hGH, at aposition corresponding to position 35 of SEQ ID NO: 2. In certainembodiments where the water-soluble polymer is a PEG, the PEG is alinear PEG. In these embodiments, the linear PEG has a MW of about 0.1to about 100 kDa, or about 1 to about 60 kDa, or about 20 to about 40kDa, or about 30 kDa. In certain embodiments encompassing a linear PEGlinked by an oxime bond to a GH, e.g., hGH, the PEG has a MW of about 30kDa. In certain embodiments where the water-soluble polymer is a PEG,the PEG is a branched PEG. In these embodiments, the branched PEG has aMW of about 1 to about 100 kDa, or about 30 to about 50 kDa, or about 40kDa. In certain embodiments encompassing a branched PEG linked by anoxime bond to a GH, e.g., hGH, the PEG has a MW of about 40 kDa.

In certain embodiments the invention provides a GH, e.g., hGH thatincludes SEQ ID NO: 2, and where the GH, e.g., hGH is substituted at aposition corresponding to position 35 of SEQ ID NO: 2 with apara-acetylphenylalanine that is linked by an oxime linkage to a linearPEG of MW of about 30 kDa.

In some embodiments, the invention provides a hormone composition thatincludes a GH, e.g., hGH, linked via an oxime bond to at least one PEG,e.g., a linear PEG, where the GH, e.g., hGH comprises the amino acidsequence of SEQ ID NO: 2, and where the GH, e.g., hGH contains at leastone non-naturally-encoded amino acid substituted at one or morepositions including, but not limited to, positions corresponding to:before position 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 8, 9, 11, 12,15, 16, 19, 22, 29, 30, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 52, 55, 57, 59, 65, 66, 69, 70, 71, 74, 88, 91,92, 94, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 111, 112, 113, 115, 116, 119, 120, 122, 123, 126, 127, 129, 130,131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144,145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 158, 159,161, 168, 172, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192 (i.e.,at the carboxyl terminus of the protein) (SEQ ID NO: 2 or thecorresponding amino acids of SEQ ID NO: 1 or 3). In some embodiments,the invention provides a hormone composition that includes a GH, e.g.,hGH, linked via an oxime bond to at least one PEG, e.g., a linear PEG,where the GH, e.g., hGH comprises the amino acid sequence of SEQ ID NO:2, and where the GH, e.g., hGH contains at least onenon-naturally-encoded amino acid substituted at one or more positionsincluding, but not limited to, positions corresponding to: 30, 35, 74,92, 103, 143, 145 (SEQ ID NO: 2 or the corresponding amino acids of SEQID NO: 1 or 3). In some embodiments, the invention provides a hormonecomposition that includes a GH, e.g., hGH, linked via an oxime bond toat least one PEG, e.g., a linear PEG, where the GH, e.g., hGH comprisesthe amino acid sequence of SEQ ID NO: 2, and where the GH, e.g., hGHcontains at least one non-naturally-encoded amino acid substituted atone or more positions including, but not limited to, positionscorresponding to: 35, 92, 143, 145 (SEQ ID NO: 2 or the correspondingamino acids of SEQ ID NO: 1 or 3). In some embodiments, the inventionprovides a hormone composition that includes a GH, e.g., hGH, linked viaan oxime bond to at least one PEG, e.g., a linear PEG, where the GH,e.g., hGH comprises the amino acid sequence of SEQ ID NO: 2, and wherethe GH, e.g., hGH contains at least one non-naturally-encoded amino acidsubstituted at one or more positions including, but not limited to,positions corresponding to: 35, 92, 131, 134, 143, 145, or anycombination thereof, from SEQ ID NO: 2 or the corresponding amino acidsof SEQ ID NO: 1 or 3. In some embodiments, the invention provides ahormone composition that includes a GH, e.g., hGH, linked via an oximebond to at least one PEG, e.g., a linear PEG, where the GH, e.g., hGHcomprises the amino acid sequence of SEQ ID NO: 2, and where the GH,e.g., hGH contains at least one non-naturally-encoded amino acidsubstituted at one or more positions including, but not limited to,positions corresponding to: 30, 35, 74, 92, 103, 145, or any combinationthereof, from SEQ ID NO: 2 or the corresponding amino acids of SEQ IDNO: 1 or 3. In some embodiments, the invention provides a hormonecomposition that includes a GH, e.g., hGH, linked via an oxime bond toat least one PEG, e.g., a linear PEG, where the GH, e.g., hGH comprisesthe amino acid sequence of SEQ ID NO: 2, and where the GH, e.g., hGHcontains at least one non-naturally-encoded amino acid substituted atone or more positions including, but not limited to, positionscorresponding to: 35, 92, 143, 145, or any combination thereof, from SEQID NO: 2 or the corresponding amino acids of SEQ ID NO: 1 or 3. In someembodiments, the invention provides a hormone composition that includesa GH, e.g., hGH, linked via an oxime bond to at least one PEG, e.g., alinear PEG, where the GH, e.g., hGH comprises the amino acid sequence ofSEQ ID NO: 2, and where the GH, e.g., hGH contains at least onenon-naturally-encoded amino acid substituted at one or more positionsincluding, but not limited to, positions corresponding to position 35from SEQ ID NO: 2 or the corresponding amino acids of SEQ ID NO: 1 or 3.In embodiments in which the PEG is a linear PEG, the PEG can have a MWof about 0.1 to about 100 kDa, or about 1 to about 60 kDa, or about 20to about 40 kDa, or about 30 kDa.

In some embodiments, the invention provides a hormone composition thatincludes a GH, e.g., hGH, linked via an oxime bond to at least one PEG,e.g., a linear PEG, where the GH, e.g., hGH includes the amino acidsequence of SEQ ID NO: 2, and where the GH, e.g., hGH contains at leastone non-naturally-encoded amino acid that is a para-acetylphenylalaninesubstituted at one or more positions including, but not limited to,positions corresponding to: before position 1 (i.e. at the N-terminus),1, 2, 3, 4, 5, 8, 9, 11, 12, 15, 16, 19, 22, 29, 30, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 52, 55, 57, 59, 65,66, 69, 70, 71, 74, 88, 91, 92, 94, 95, 97, 98, 99, 100, 101, 102, 103,104, 105, 106, 107, 108, 109, 111, 112, 113, 115, 116, 119, 120, 122,123, 126, 127, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139,140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153,154, 155, 156, 158, 159, 161, 168, 172, 183, 184, 185, 186, 187, 188,189, 190, 191, 192 (i.e., at the carboxyl terminus of the protein) (SEQID NO: 2 or the corresponding amino acids of SEQ ID NO: 1 or 3). In someembodiments, the invention provides a hormone composition that includesa GH, e.g., hGH, linked via an oxime bond to at least one PEG, e.g., alinear PEG, where the GH, e.g., hGH comprises the amino acid sequence ofSEQ ID NO: 2, and where the GH, e.g., hGH contains at least onenon-naturally-encoded amino acid that is a para-acetylphenylalaninesubstituted at one or more positions including, but not limited to,positions corresponding to: 30, 35, 74, 92, 103, 143, 145 (SEQ ID NO: 2or the corresponding amino acids of SEQ ID NO: 1 or 3). In someembodiments, the invention provides a hormone composition that includesa GH, e.g., hGH, linked via an oxime bond to at least one PEG, e.g., alinear PEG, where the GH, e.g., hGH comprises the amino acid sequence ofSEQ ID NO: 2, and where the GH, e.g., hGH contains at least onenon-naturally-encoded amino acid that is a para-acetylphenylalaninesubstituted at one or more positions including, but not limited to,positions corresponding to: 35, 92, 143, 145 (SEQ ID NO: 2 or thecorresponding amino acids of SEQ ID NO: 1 or 3). In some embodiments,the invention provides a hormone composition that includes a GH, e.g.,hGH, linked via an oxime bond to at least one PEG, e.g., a linear PEG,where the GH, e.g., hGH comprises the amino acid sequence of SEQ. ID NO:2, and where the GH, e.g., hGH contains at least onenon-naturally-encoded amino acid that is a para-acetylphenylalaninesubstituted at one or more positions including, but not limited to,positions corresponding to: 35, 92, 131, 134, 143, 145, or anycombination thereof, from SEQ ID NO: 2 or the corresponding amino acidsof SEQ ID NO: 1 or 3. In some embodiments, the invention provides ahormone composition that includes a GH, e.g., hGH, linked via an oximebond to at least one PEG, e.g., a linear PEG, where the GH, e.g., hGHcomprises the amino acid sequence of SEQ ID NO: 2, and where the GH,e.g., hGH contains at least one non-naturally-encoded amino acid that isa para-acetylphenylalanine substituted at one or more positionsincluding, but not limited to, positions corresponding to: 30, 35, 74,92, 103, 145, or any combination thereof, from SEQ ID NO: 2 or thecorresponding amino acids of SEQ ID NO: 1 or 3. In some embodiments, theinvention provides a hormone composition that includes a GH, e.g., hGH,linked via an oxime bond to at least one PEG, e.g., a linear PEG, wherethe GH, e.g., hGH comprises the amino acid sequence of SEQ ID NO: 2, andwhere the GH, e.g., hGH contains at least one non-naturally-encodedamino acid that is a para-acetylphenylalanine substituted at one or morepositions including, but not limited to, positions corresponding to: 35,92, 143, 145, or any combination thereof, from SEQ ID NO: 2 or thecorresponding amino acids of SEQ ID NO: 1 or 3. In some embodiments, theinvention provides a hormone composition that includes a GH, e.g., hGH,linked via an oxime bond to at least one PEG, e.g., a linear PEG, wherethe GH, e.g., hGH comprises the amino acid sequence of SEQ ID NO: 2, andwhere the GH, e.g., hGH contains at least one non-naturally-encodedamino acid that is a para-acetylphenylalanine substituted at one or morepositions including, but not limited to, positions corresponding toposition 35 from SEQ ID NO: 2 or the corresponding amino acids of SEQ IDNO: 1 or 3. In embodiments in which the PEG is a linear PEG, the PEG canhave a MW of about 0.1 to about 100 kDa, or about 1 to about 60 kDa, orabout 20 to about 40 kDa, or about 30 kDa.

In some embodiments, the invention provides a GH, e.g., hGH, where theGH, e.g., hGH contains at least one non-naturally encoded amino acid,where the GH is linked to a plurality of water-soluble polymers, e.g., aplurality of PEGs, by covalent bonds, where one or more of the covalentbond is an oxime bond between at least one of the non-naturally encodedamino acid and the water-soluble polymer, e.g., PEG. The GH, e.g., hGH,may be linked to about 2-100 water-soluble polymers, e.g., PEGs, orabout 2-50 water-soluble polymers, e.g., PEGs, or about 2-25water-soluble polymers, e.g., PEGs, or about 2-10 water-solublepolymers, e.g., PEGs, or about 2-5 water-soluble polymers, e.g., PEGs,or about 5-100 water-soluble polymers, e.g., PEGs, or about 5-50water-soluble polymers, e.g., PEGs, or about 5-25 water-solublepolymers, e.g., PEGs, or about 5-10 water-soluble polymers, e.g., PEGs,or about 10-100 water-soluble polymers, e.g., PEGs, or about 10-50water-soluble polymers, e.g., PEGs, or about 10-20 water-solublepolymers, e.g., PEGs, or about 20-100 water-soluble polymers, e.g.,PEGs, or about 20-50 water-soluble polymers, e.g., PEGs, or about 50-100water-soluble polymers, e.g., PEGs. The one or morenon-naturally-encoded amino acids may be incorporated into the GH, e.g.,hGH, at any position described herein. In some embodiments, at least onenon-naturally-encoded amino acid is incorporated into the GH, e.g., hGH,at a position corresponding to position 35 of SEQ ID NO: 2. In someembodiments, the non-naturally encoded amino acids include at least onenon-naturally encoded amino acid that is a carbonyl-containingnon-naturally encoded amino acid, e.g., a ketone-containingnon-naturally encoded amino acid such as a para-acetylphenylalanine. Insome embodiments, the GH, e.g., hGH, includes apara-acetylphenylalanine. In some embodiments, thepara-acetylphenylalanine is incorporated into the GH, e.g., hGH, at aposition corresponding to position 35 of SEQ ID NO: 2, where thepara-acetylphenylalanine is linked to one of the polymers, e.g., one ofthe PEGs, by an oxime bond. In some embodiments, at least one of thewater-soluble polymers, e.g., PEGs, is linked to the GH, e.g., hGH, by acovalent bond to at least one of the non-naturally-encoded amino acids.In some embodiments, the covalent bond is an oxime bond. In someembodiments, a plurality of the water-soluble polymers, e.g., PEGs, arelinked to the GH, e.g., hGH, by covalent bonds to a plurality of thenon-naturally-encoded amino acids. In some embodiments, at least one thecovalent bonds is an oxime bond; in some embodiments, a plurality of thecovalent bonds are oxime bonds; in some embodiments, substantially allof the bonds are oxime bonds. The plurality of water-soluble polymers,e.g., PEG, may be linear, branched, or any combination thereof. Inembodiments that incorporate one or more linear PEGs, the linear PEGshave a MW of about 0.1 to about 100 kDa, or about 1 to about 60 kDa, orabout 20 to about 40 kDa, or about 30 kDa. In embodiments thatincorporate one or more branched PEGs, the branched PEGs have a MW ofabout 1 to about 100 kDa, or about 30 to about 50 kDa, or about 40 kDa.It will be appreciated that embodiments employing a plurality ofwater-soluble polymers, e.g., PEGs, will, in general, employ suchpolymers at lower MWs than embodiments in which a single PEG is used.Thus, in some embodiments, the overall MW of the plurality of PEGs isabout 0.1-500 kDa, or about 0.1-200 kDa, or about 0.1-100 kDa, or about1-1000 kDa, or about 1-500 kDa, or about 1-200 kDa, or about 1-100 kDa,or about 10-1000 kDa, or about 10-500 kDa, or about 10-200 kDa, or about10-100 kDa, or about 10-50 kDa, or about 20-1000 kDa, or about 20-500kDa, or about 20-200 kDa, or about 20-100 kDa, or about 20-80 kDa, about20-60 kDa, about 5-100 kDa, about 5-50 kDa, or about 5-20 kDa.

Human GH antagonists include, but are not limited to, those withsubstitutions at: 1, 2, 3, 4, 5, 8, 9, 11, 12, 15, 16, 19, 22, 103, 109,112, 113, 115, 116, 119, 120, 123, and 127 or an addition at position 1(i.e., at the N-terminus), or any combination thereof (SEQ ID NO:2, orthe corresponding amino acid in SEQ ID NO: 1, 3, or any other GHsequence).

A wide variety of non-naturally encoded amino acids can be substitutedfor, or incorporated into, a given position in a GH, e.g., hGHpolypeptide. In general, a particular non-naturally encoded amino acidis selected for incorporation based on an examination of the threedimensional crystal structure of a GH, e.g., hGH polypeptide with itsreceptor, a preference for conservative substitutions (i.e., aryl-basednon-naturally encoded amino acids, such as p-acetylphenylalanine orO-propargyltyrosine substituting for Phe, Tyr or Trp), and the specificconjugation chemistry that one desires to introduce into the GH, e.g.,hGH polypeptide (e.g., the introduction of 4-azidophenylalanine if onewants to effect a Huisgen [3+2]cycloaddition with a water solublepolymer bearing an alkyne moiety or a amide bond formation with a watersoluble polymer that bears an aryl ester that, in turn, incorporates aphosphine moiety).

In one embodiment, the method further includes incorporating into theprotein the unnatural amino acid, where the unnatural amino acidcomprises a first reactive group; and contacting the protein with amolecule (including but not limited to, a label, a dye, a polymer, awater-soluble polymer, a derivative of polyethylene glycol, aphotocrosslinker, a radionuclide, a cytotoxic compound, a drug, anaffinity label, a photoaffinity label, a reactive compound, a resin, asecond protein or polypeptide or polypeptide analog, an antibody orantibody fragment, a metal chelator, a cofactor, a fatty acid, acarbohydrate, a polynucleotide, a DNA, a RNA, an antisensepolynucleotide, a saccharide, water-soluble dendrimer, a cyclodextrin,an inhibitory ribonucleic acid, a biomaterial, a nanoparticle, a spinlabel, a fluorophore, a metal-containing moiety, a radioactive moiety, anovel functional group, a group that covalently or noncovalentlyinteracts with other molecules, a photocaged moiety, an actinicradiation excitable moiety, a photoisomerizable moiety, biotin, aderivative of biotin, a biotin analogue, a moiety incorporating a heavyatom, a chemically cleavable group, a photocleavable group, an elongatedside chain, a carbon-linked sugar, a redox-active agent, an aminothioacid, a toxic moiety, an isotopically labeled moiety, a biophysicalprobe, a phosphorescent group, a chemiluminescent group, an electrondense group, a magnetic group, an intercalating group, a chromophore, anenergy transfer agent, a biologically active agent, a detectable label,a small molecule, a quantum dot, a nanotransmitter, a radionucleotide, aradiotransmitter, a neutron-capture agent, or any combination of theabove, or any other desirable compound or substance) that comprises asecond reactive group. The first reactive group reacts with the secondreactive group to attach the molecule to the unnatural amino acidthrough a [3+2]cycloaddition. In one embodiment, the first reactivegroup is an alkynyl or azido moiety and the second reactive group is anazido or alkynyl moiety. For example, the first reactive group is thealkynyl moiety (including but not limited to, in unnatural amino acidp-propargyloxyphenylalanine) and the second reactive group is the azidomoiety. In another example, the first reactive group is the azido moiety(including but not limited to, in the unnatural amino acidp-azido-L-phenylalanine) and the second reactive group is the alkynylmoiety.

In some cases, the non-naturally encoded amino acid substitution(s) willbe combined with other additions, substitutions or deletions within theGH, e.g., hGH polypeptide to affect other biological traits of the GH,e.g., hGH polypeptide. In some cases, the other additions, substitutionsor deletions may increase the stability (including but not limited to,resistance to proteolytic degradation) of the GH, e.g., hGH polypeptideor increase affinity of the GH, e.g., hGH polypeptide for its receptor.In some embodiments, the GH, e.g., hGH polypeptide comprises an aminoacid substitution selected from the group consisting of F10A, F10H,F10I; M14W, M14Q, M14G; H18D; H21N; G120A; R167N; D171S; E174S; F176Y,I179T or any combination thereof in SEQ ID NO: 2. In some cases, theother additions, substitutions or deletions may increase the solubility(including but not limited to, when expressed in E. coli or other hostcells) of the GH, e.g., hGH polypeptide. In some embodiments additions,substitutions or deletions may increase the polypeptide solubilityfollowing expression in E. coli or other recombinant host cells. In someembodiments sites are selected for substitution with a naturally encodedor non-natural amino acid in addition to another site for incorporationof a non-natural amino acid that results in increasing the polypeptidesolubility following expression in E. coli or other recombinant hostcells. In some embodiments, the GH, e.g., hGH polypeptides compriseanother addition, substitution or deletion that modulates affinity forthe GH, e.g., hGH polypeptide receptor, modulates (including but notlimited to, increases or decreases) receptor dimerization, stabilizesreceptor dimers, modulates circulating half-life, modulates release orbio-availability, facilitates purification, or improves or alters aparticular route of administration. For instance, in addition tointroducing one or more non-naturally encoded amino acids as set forthherein, one or more of the following substitutions are introduced: F10A,F10H or F10I; M14W, M14Q, or M14G; H18D; H21N; R167N; D171S; E174S;F176Y and I179T to increase the affinity of the GH, e.g., hGH variantfor its receptor. Similarly, GH, e.g., hGH polypeptides can comprisechemical or enzyme cleavage sequences, protease cleavage sequences,reactive groups, antibody-binding domains (including but not limited to,FLAG or poly-His) or other affinity based sequences (including, but notlimited to, FLAG, poly-His, GST, etc.) or linked molecules (including,but not limited to, biotin) that improve detection (including, but notlimited to, GFP), purification, transport through tissues or cellmembranes, prodrug release or activation, hGH size reduction, or othertraits of the polypeptide.

In some embodiments, the substitution of a non-naturally encoded aminoacid generates an GH, e.g., hGH antagonist. A subset of exemplary sitesfor incorporation of one or more non-naturally encoded amino acidinclude: 1, 2, 3, 4, 5, 8, 9, 11, 12, 15, 16, 19, 22, 103, 109, 112,113, 115, 116, 119, 120, 123, 127, or an addition before position 1 (SEQID NO: 2, or the corresponding amino acid in SEQ ID NO: 1, 3, or anyother GH sequence). In some embodiments, GH, e.g., hGH antagonistscomprise at least one substitution in the regions 1-5 (N-terminus), 6-33(A helix), 34-74 (region between A helix and B helix, the A-B loop),75-96 (B helix), 97-105 (region between B helix and C helix, the B-Cloop), 106-129 (C helix), 130-153 (region between C helix and D helix,the C-D loop), 154-183 (D helix), 184-191 (C-terminus) that cause GH toact as an antagonist. In other embodiments, the exemplary sites ofincorporation of a non-naturally encoded amino acid include residueswithin the amino terminal region of helix A and a portion of helix C. Inanother embodiment, substitution of G120 with a non-naturally encodedamino acid such as p-azido-L-phenyalanine or O-propargyl-L-tyrosine. Inother embodiments, the above-listed substitutions are combined withadditional substitutions that cause the GH, e.g., hGH polypeptide to bean GH, e.g., hGH antagonist. For instance, a non-naturally encoded aminoacid is substituted at one of the positions identified herein and asimultaneous substitution is introduced at G120 (e.g., G120R, G120K,G120W, G120Y, G120F, or G120E). In some embodiments, the GH, e.g., hGHantagonist comprises a non-naturally encoded amino acid linked to awater soluble polymer that is present in a receptor binding region ofthe GH, e.g., hGH molecule.

In some cases, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids aresubstituted with one or more non-naturally-encoded amino acids. In somecases, the GH, e.g., hGH polypeptide further includes 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or more substitutions of one or more non-naturally encodedamino acids for naturally-occurring amino acids. For example, in someembodiments, one or more residues in the following regions of GH, e.g.,hGH are substituted with one or more non-naturally encoded amino acids:1-5 (N-terminus); 32-46 (N-terminal end of the A-B loop); 97-105 (B-Cloop); and 132-149 (C-D loop); and 184-191 (C-terminus). In someembodiments, one or more residues in the following regions of GH, e.g.,hGH are substituted with one or more non-naturally encoded amino acids:1-5 (N-terminus), 6-33 (A helix), 34-74 (region between A helix and Bhelix, the A-B loop), 75-96 (B helix), 97-105 (region between B helixand C helix, the B-C loop), 106-129 (C helix), 130-153 (region between Chelix and D helix, the C-D loop), 154-183 (D helix), 184-191(C-terminus). In some cases, the one or more non-naturally encodedresidues are linked to one or more lower molecular weight linear orbranched PEGs (approximately ˜5-20 kDa in mass or less), therebyenhancing binding affinity and comparable serum half-life relative tothe species attached to a single, higher molecular weight PEG.

In some embodiments, up to two of the following residues of GH, e.g.,hGH are substituted with one or more non-naturally-encoded amino acidsat position: 29, 30, 33, 34, 35, 37, 39, 40, 49, 57, 59, 66, 69, 70, 71,74, 88, 91, 92, 94, 95, 98, 99, 101, 103, 107, 108, 111, 122, 126, 129,130, 131, 133, 134, 135, 136, 137, 139, 140, 141, 142, 143, 145, 147,154, 155, 156, 159, 183, 186, and 187. In some cases, any of thefollowing pairs of substitutions are made: K38X* and K140X*; K41X* andK145X*; Y35X* and E88X*; Y35X* and F92X*; Y35X* and Y143X*; F92X* andY143X* wherein X* represents a non-naturally encoded amino acid.Preferred sites for incorporation of two or more non-naturally encodedamino acids include combinations of the following residues: 29, 33, 35,37, 39, 49, 57, 69, 70, 71, 74, 88, 91, 92, 94, 95, 98, 99, 101, 103,107, 108, 111, 129, 130, 131, 133, 134, 135, 136, 137, 139, 140, 141,142, 143, 145, 147, 154, 155, 156, 186, and 187. Particularly preferredsites for incorporation of two or more non-naturally encoded amino acidsinclude combinations of the following residues: 35, 88, 91, 92, 94, 95,99, 101, 103, 111, 131, 133, 134, 135, 136, 139, 140, 143, 145, and 155.

Preferred sites for incorporation in GH, e.g., hGH of two or morenon-naturally encoded amino acids include combinations of the followingresidues: before position 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 8,9, 11, 12, 15, 16, 19, 22, 29, 30, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 52, 55, 57, 59, 65, 66, 69, 70, 71,74, 88, 91, 92, 94, 95, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106,107, 108, 109, 111, 112, 113, 115, 116, 119, 120, 122, 123, 126, 127,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,158, 159, 161, 168, 172, 183, 184, 185, 186, 187, 188, 189, 190, 191,192 (i.e. at the carboxyl terminus of the protein) or any combinationthereof from SEQ ID NO: 2.

VII. Expression in Non-Eukaryotes and Eukaryotes

To obtain high level expression of a cloned GH, e.g., hGHpolynucleotide, one typically subclones polynucleotides encoding a GH,e.g., hGH polypeptide of the invention into an expression vector thatcontains a strong promoter to direct transcription, atranscription/translation terminator, and if for a nucleic acid encodinga protein, a ribosome binding site for translational initiation.Suitable bacterial promoters are known to those of ordinary skill in theart and described, e.g., in Sambrook et al. and Ausubel et al.

Bacterial expression systems for expressing GH, e.g., hGH polypeptidesof the invention are available in, including but not limited to, E.coli, Bacillus sp., Pseudomonas fluorescens, Pseudomonas aeruginosa,Pseudomonas putida, and Salmonella (Palva et al., Gene 22:229-235(1983); Mosbach et al., Nature 302:543-545 (1983)). Kits for suchexpression systems are commercially available. Eukaryotic expressionsystems for mammalian cells, yeast, and insect cells are known to thoseof ordinary skill in the art and are also commercially available. Incases where orthogonal tRNAs and aminoacyl tRNA synthetases (describedabove) are used to express the GH, e.g., hGH polypeptides of theinvention, host cells for expression are selected based on their abilityto use the orthogonal components. Exemplary host cells includeGram-positive bacteria (including but not limited to B. brevis, B.subtilis, or Streptomyces) and Gram-negative bacteria (E. coli,Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas putida), aswell as yeast and other eukaryotic cells. Cells comprising O-tRNA/O—RSpairs can be used as described herein.

A eukaryotic host cell or non-eukaryotic host cell of the presentinvention provides the ability to synthesize proteins that compriseunnatural amino acids in large useful quantities. In one aspect, thecomposition optionally includes, including but not limited to, at least10 micrograms, at least 50 micrograms, at least 75 micrograms, at least100 micrograms, at least 200 micrograms, at least 250 micrograms, atleast 500 micrograms, at least 1 milligram, at least 10 milligrams, atleast 100 milligrams, at least one gram, or more of the protein thatcomprises an unnatural amino acid, or an amount that can be achievedwith in vivo protein production methods (details on recombinant proteinproduction and purification are provided herein). In another aspect, theprotein is optionally present in the composition at a concentration of,including but not limited to, at least 10 micrograms of protein perliter, at least 50 micrograms of protein per liter, at least 75micrograms of protein per liter, at least 100 micrograms of protein perliter, at least 200 micrograms of protein per liter, at least 250micrograms of protein per liter, at least 500 micrograms of protein perliter, at least 1 milligram of protein per liter, or at least 10milligrams of protein per liter or more, in, including but not limitedto, a cell lysate, a buffer, a pharmaceutical buffer, or other liquidsuspension (including but not limited to, in a volume of, including butnot limited to, anywhere from about 1 nl to about 100 L). The productionof large quantities (including but not limited to, greater that thattypically possible with other methods, including but not limited to, invitro translation) of a protein in a eukaryotic cell including at leastone unnatural amino acid is a feature of the invention.

A eukaryotic host cell or non-eukaryotic host cell of the presentinvention provides the ability to biosynthesize proteins that compriseunnatural amino acids in large useful quantities. For example, proteinscomprising an unnatural amino acid can be produced at a concentrationof, including but not limited to, at least 10 μg/liter, at least 50μg/liter, at least 75 μg/liter, at least 100 μg/liter, at least 200μg/liter, at least 250 μg/liter, or at least 500 μg/liter, at least 1mg/liter, at least 2 mg/liter, at least 3 mg/liter, at least 4 mg/liter,at least 5 mg/liter, at least 6 mg/liter, at least 7 mg/liter, at least8 mg/liter, at least 9 mg/liter, at least 10 mg/liter, at least 20, 30,40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900mg/liter, 1 g/liter, 5 g/liter, 10 g/liter or more of protein in a cellextract, cell lysate, culture medium, a buffer, and/or the like.

I. Expression Systems, Culture, and Isolation

GH, e.g., hGH polypeptides may be expressed in any number of suitableexpression systems including, for example, yeast, insect cells,mammalian cells, and bacteria. A description of exemplary expressionsystems is provided below.

Yeast As used herein, the term “yeast” includes any of the variousyeasts capable of expressing a gene encoding a GH, e.g., hGHpolypeptide. Such yeasts include, but are not limited to,ascosporogenous yeasts (Endomycetales), basidiosporogenous yeasts andyeasts belonging to the Fungi imperfecti (Blastomycetes) group. Theascosporogenous yeasts are divided into two families, Spermophthoraceaeand Saccharomycetaceae. The latter is comprised of four subfamilies,Schizosaccharomycoideae (e.g., genus Schizosaccharomyces),Nadsonioideae, Lipomycoideae and Saccharomycoideae (e.g., genera Pichia,Kluyveromyces and Saccharomyces). The basidiosporogenous yeasts includethe genera Leucosporidium, Rhodosporidium, Sporidiobolus, Filobasidium,and Filobasidiella. Yeasts belonging to the Fungi Imperfecti(Blastomycetes) group are divided into two families, Sporobolomycetaceae(e.g., genera Sporobolomyces and Bullera) and Cryptococcaceae (e.g.,genus Candida).

Of particular interest for use with the present invention are specieswithin the genera Pichia, Kluyveromyces, Saccharomyces,Schizosaccharomyces, Hansenula, Torulopsis, and Candida, including, butnot limited to, P. pastoris, P. guillerimondii, S. cerevisiae, S.carlsbergensis, S. diastaticus, S. douglasii, S. kluyveri, S, norbensis,S. oviformis, K. lactis, K., fragilis, C. albicans, C. maltosa, and H.polymorpha.

The selection of suitable yeast for expression of GH, e.g., hGHpolypeptides is within the skill of one of ordinary skill in the art. Inselecting yeast hosts for expression, suitable hosts may include thoseshown to have, for example, good secretion capacity, low proteolyticactivity, good secretion capacity, good soluble protein production, andoverall robustness. Yeast are generally available from a variety ofsources including, but not limited to, the Yeast Genetic Stock Center,Department of Biophysics and Medical Physics, University of California(Berkeley, Calif.), and the American Type Culture Collection (“ATCC”)(Manassas, Va.).

The term “yeast host” or “yeast host cell” includes yeast that can be,or has been, used as a recipient for recombinant vectors or othertransfer DNA. The term includes the progeny of the original yeast hostcell that has received the recombinant vectors or other transfer DNA. Itis understood that the progeny of a single parental cell may notnecessarily be completely identical in morphology or in genomic or totalDNA complement to the original parent, due to accidental or deliberatemutation. Progeny of the parental cell that are sufficiently similar tothe parent to be characterized by the relevant property, such as thepresence of a nucleotide sequence encoding a GH, e.g., hGH polypeptide,are included in the progeny intended by this definition.

Expression and transformation vectors, including extrachromosomalreplicons or integrating vectors, have been developed for transformationinto many yeast hosts. For example, expression vectors have beendeveloped for S. cerevisiae (Sikorski et al., GENETICS (1989) 122:19;Ito et al., J. BACTERIOL. (1983) 153:163; Hinnen et al., PROC. NATL.ACAD. SCI. USA (1978) 75:1929); C. albicans (Kurtz et al., MOL. CELL.BIOL. (1986) 6:142); C. maltosa (Kunze et al., J. BASIC MICROBIOL.(1985) 25:141); H. polymorpha (Gleeson et al., J. GEN. MICROBIOL. (1986)132:3459; Roggenkamp et al., MOL. GENETICS AND GENOMICS (1986) 202:302);K. fragilis (Das et al., J. BACTERIOL. (1984) 158:1165); K. lactis (DeLouvencourt et al., J. BACTERIOL. (1983) 154:737; Van den Berg et al.,BIO TECHNOLOGY (NY) (1990) 8:135); P. guillerimondii (Kunze et al., J.BASIC MICROBIOL. (1985) 25:141); P. pastoris (U.S. Pat. Nos. 5,324,639;4,929,555; and 4,837,148; Cregg et al., MOL. CELL. BIOL. (1985) 5:3376);Schizosaccharomyces pombe (Beach et al., NATURE (1982) 300:706); and Y.lipolytica; A. nidulans (Ballance et al., BIOCHEM. BIOPHYS. RES. COMMUN.(1983) 112:284-89; Tilburn et al., GENE (1983) 26:205-221; and Yelton etal., PROC. NATL. ACAD. SCI. USA (1984) 81:1470-74); A. niger (Kelly andHynes, EMBO J. (1985) 4:475-479); T. reesia (EP 0 244 234); andfilamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium(WO 91/00357), each incorporated by reference herein.

Control sequences for yeast vectors are known to those of ordinary skillin the art and include, but are not limited to, promoter regions fromgenes such as alcohol dehydrogenase (ADH) (EP 0 284 044); enolase;glucokinase; glucose-6-phosphate isomerase;glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH); hexokinase;phosphofructokinase; 3-phosphoglycerate mutase; and pyruvate kinase(PyK) (EP 0 329 203). The yeast PHO5 gene, encoding acid phosphatase,also may provide useful promoter sequences (Miyanohara et al., PROC.NATL. ACAD. SCI. USA (1983) 80:1). Other suitable promoter sequences foruse with yeast hosts may include the promoters for 3-phosphoglyceratekinase (Hitzeman et al., J. BIOL. CHEM. (1980) 255:12073); and otherglycolytic enzymes, such as pyruvate decarboxylase, triosephosphateisomerase, and phosphoglucose isomerase (Holland et al., BIOCHEMISTRY(1978) 17:4900; Hess et al., J. ADV. ENZYME REG. (1969) 7:149).Inducible yeast promoters having the additional advantage oftranscription controlled by growth conditions may include the promoterregions for alcohol dehydrogenase 2; isocytochrome C; acid phosphatase;metallothionein; glyceraldehyde-3-phosphate dehydrogenase; degradativeenzymes associated with nitrogen metabolism; and enzymes responsible formaltose and galactose utilization. Suitable vectors and promoters foruse in yeast expression are further described in EP 0 073 657.

Yeast enhancers also may be used with yeast promoters. In addition,synthetic promoters may also function as yeast promoters. For example,the upstream activating sequences (UAS) of a yeast promoter may bejoined with the transcription activation region of another yeastpromoter, creating a synthetic hybrid promoter. Examples of such hybridpromoters include the ADH regulatory sequence linked to the GAPtranscription activation region. See U.S. Pat. Nos. 4,880,734 and4,876,197, which are incorporated by reference herein. Other examples ofhybrid promoters include promoters that consist of the regulatorysequences of the ADH2, GAL4, GAL10, or PHO5 genes, combined with thetranscriptional activation region of a glycolytic enzyme gene such asGAP or PyK. See EP 0 164 556. Furthermore, a yeast promoter may includenaturally occurring promoters of non-yeast origin that have the abilityto bind yeast RNA polymerase and initiate transcription.

Other control elements that may comprise part of the yeast expressionvectors include terminators, for example, from GAPDH or the enolasegenes (Holland et al., J. BIOL. CHEM. (1981) 256:1385). In addition, theorigin of replication from the 2/plasmid origin is suitable for yeast. Asuitable selection gene for use in yeast is the trp1 gene present in theyeast plasmid. See Tschumper et al., GENE (1980) 10:157; Kingsman etal., GENE (1979) 7:141. The trp1 gene provides a selection marker for amutant strain of yeast lacking the ability to grow in tryptophan.Similarly, Leu2-deficient yeast strains (ATCC 20,622 or 38,626) arecomplemented by known plasmids bearing the Leu2 gene.

Methods of introducing exogenous DNA into yeast hosts are known to thoseof ordinary skill in the art, and typically include, but are not limitedto, either the transformation of spheroplasts or of intact yeast hostcells treated with alkali cations. For example, transformation of yeastcan be carried out according to the method described in Hsiao et al.,PROC. NATL. ACAD. SCI. USA (1979) 76:3829 and Van Solingen et al., J.BACT. (1977) 130:946. However, other methods for introducing DNA intocells such as by nuclear injection, electroporation, or protoplastfusion may also be used as described generally in SAMBROOK ET AL.,MOLECULAR CLONING: A LAB. MANUAL (2001). Yeast host cells may then becultured using standard techniques known to those of ordinary skill inthe art.

Other methods for expressing heterologous proteins in yeast host cellsare known to those of ordinary skill in the art. See generally U.S.Patent Publication No. 20020055169, U.S. Pat. Nos. 6,361,969; 6,312,923;6,183,985; 6,083,723; 6,017,731; 5,674,706; 5,629,203; 5,602,034; and5,089,398; U.S. Reexamined Patent Nos. RE37,343 and RE35,749; PCTPublished Patent Applications WO 99/07862; WO 98/37208; and WO 98/26080;European Patent Applications EP 0 946 736; EP 0 732 403; EP 0 480 480;WO 90/10277; EP 0 340 986; EP 0 329 203; EP 0 324 274; and EP 0 164 556.See also Gellissen et al., ANTONIE VAN LEEUWENHOEK (1992) 62(1-2):79-93;Romanos et al., YEAST (1992) 8(6):423-488; Goeddel, METHODS INENZYMOLOGY (1990) 185:3-7, each incorporated by reference herein.

The yeast host strains may be grown in fermentors during theamplification stage using standard feed batch fermentation methods knownto those of ordinary skill in the art. The fermentation methods may beadapted to account for differences in a particular yeast host's carbonutilization pathway or mode of expression control. For example,fermentation of a Saccharomyces yeast host may require a single glucosefeed, complex nitrogen source (e.g., casein hydrolysates), and multiplevitamin supplementation. In contrast, the methylotrophic yeast P.pastoris may require glycerol, methanol, and trace mineral feeds, butonly simple ammonium (nitrogen) salts for optimal growth and expression.See, e.g., U.S. Pat. No. 5,324,639; Elliott et al., J. PROTEIN CHEM.(1990) 9:95; and Fieschko et al., BIOTECH. BIOENG. (1987) 29:1113,incorporated by reference herein.

Such fermentation methods, however, may have certain common featuresindependent of the yeast host strain employed. For example, a growthlimiting nutrient, typically carbon, may be added to the fermentorduring the amplification phase to allow maximal growth. In addition,fermentation methods generally employ a fermentation medium designed tocontain adequate amounts of carbon, nitrogen, basal salts, phosphorus,and other minor nutrients (vitamins, trace minerals and salts, etc.).Examples of fermentation media suitable for use with Pichia aredescribed in U.S. Pat. Nos. 5,324,639 and 5,231,178, which areincorporated by reference herein.

Baculovirus-Infected Insect Cells The term “insect host” or “insect hostcell” refers to a insect that can be, or has been, used as a recipientfor recombinant vectors or other transfer DNA. The term includes theprogeny of the original insect host cell that has been transfected. Itis understood that the progeny of a single parental cell may notnecessarily be completely identical in morphology or in genomic or totalDNA complement to the original parent, due to accidental or deliberatemutation. Progeny of the parental cell that are sufficiently similar tothe parent to be characterized by the relevant property, such as thepresence of a nucleotide sequence encoding a GH, e.g., hGH polypeptide,are included in the progeny intended by this definition.

The selection of suitable insect cells for expression of GH, e.g., hGHpolypeptides is known to those of ordinary skill in the art. Severalinsect species are well described in the art and are commerciallyavailable including Aedes aegypti, Bombyx mori, Drosophila melanogaster,Spodoptera frugiperda, and Trichoplusia ni. In selecting insect hostsfor expression, suitable hosts may include those shown to have, interalia, good secretion capacity, low proteolytic activity, and overallrobustness. Insect are generally available from a variety of sourcesincluding, but not limited to, the Insect Genetic Stock Center,Department of Biophysics and Medical Physics, University of California(Berkeley, Calif.); and the American Type Culture Collection (“ATCC”)(Manassas, Va.).

Generally, the components of a baculovirus-infected insect expressionsystem include a transfer vector, usually a bacterial plasmid, whichcontains both a fragment of the baculovirus genome, and a convenientrestriction site for insertion of the heterologous gene to be expressed;a wild type baculovirus with sequences homologous to thebaculovirus-specific fragment in the transfer vector (this allows forthe homologous recombination of the heterologous gene in to thebaculovirus genome); and appropriate insect host cells and growth media.The materials, methods and techniques used in constructing vectors,transfecting cells, picking plaques, growing cells in culture, and thelike are known in the art and manuals are available describing thesetechniques.

After inserting the heterologous gene into the transfer vector, thevector and the wild type viral genome are transfected into an insecthost cell where the vector and viral genome recombine. The packagedrecombinant virus is expressed and recombinant plaques are identifiedand purified. Materials and methods for baculovirus/insect cellexpression systems are commercially available in kit form from, forexample, Invitrogen Corp. (Carlsbad, Calif.). These techniques aregenerally known to those of ordinary skill in the art and fullydescribed in SUMMERS AND SMITH, TEXAS AGRICULTURAL EXPERIMENT STATIONBULLETIN NO. 1555 (1987), herein incorporated by reference. See also,RICHARDSON, 39 METHODS IN MOLECULAR BIOLOGY: BACULOVIRUS EXPRESSIONPROTOCOLS (1995); AUSUBEL ET AL., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY16.9-16.11 (1994); KING AND POSSEE, THE BACULOVIRUS SYSTEM: A LABORATORYGUIDE (1992); and O'REILLY ET AL., BACULOVIRUS EXPRESSION VECTORS: ALABORATORY MANUAL (1992).

Indeed, the production of various heterologous proteins usingbaculovirus/insect cell expression systems is known to those of ordinaryskill in the art. See, e.g., U.S. Pat. Nos. 6,368,825; 6,342,216;6,338,846; 6,261,805; 6,245,528, 6,225,060; 6,183,987; 6,168,932;6,126,944; 6,096,304; 6,013,433; 5,965,393; 5,939,285; 5,891,676;5,871,986; 5,861,279; 5,858,368; 5,843,733; 5,762,939; 5,753,220;5,605,827; 5,583,023; 5,571,709; 5,516,657; 5,290,686; WO 02/06305; WO01/90390; WO 01/27301; WO 01/05956; WO 00/55345; WO 00/20032; WO99/51721; WO 99/45130; WO 99/31257; WO 99/10515; WO 99/09193; WO97/26332; WO 96/29400; WO 96/25496; WO 96/06161; WO 95/20672; WO93/03173; WO 92/16619; WO 92/02628; WO 92/01801; WO 90/14428; WO90/10078; WO 90/02566; WO 90/02186; WO 90/01556; WO 89/01038; WO89/01037; WO 88/07082, which are incorporated by reference herein.

Vectors that are useful in baculovirus/insect cell expression systemsare known in the art and include, for example, insect expression andtransfer vectors derived from the baculovirus Autographacalifornicanuclear polyhedrosis virus (AcNPV), which is a helper-independent, viralexpression vector. Viral expression vectors derived from this systemusually use the strong viral polyhedrin gene promoter to driveexpression of heterologous genes. See generally, O'Reilly ET AL.,BACULOVIRUS EXPRESSION VECTORS: A LABORATORY MANUAL (1992).

Prior to inserting the foreign gene into the baculovirus genome, theabove-described components, comprising a promoter, leader (if desired),coding sequence of interest, and transcription termination sequence, aretypically assembled into an intermediate transplacement construct(transfer vector). Intermediate transplacement constructs are oftenmaintained in a replicon, such as an extra chromosomal element (e.g.,plasmids) capable of stable maintenance in a host, such as bacteria. Thereplicon will have a replication system, thus allowing it to bemaintained in a suitable host for cloning and amplification. Morespecifically, the plasmid may contain the polyhedrin polyadenylationsignal (Miller, ANN. REV. MICROBIOL. (1988) 42:177) and a prokaryoticampicillin-resistance (amp) gene and origin of replication for selectionand propagation in E. coli.

One commonly used transfer vector for introducing foreign genes intoAcNPV is pAc373. Many other vectors, known to those of skill in the art,have also been designed including, for example, pVL985, which alters thepolyhedrin start codon from ATG to ATT, and which introduces a BamHIcloning site 32 base pairs downstream from the ATT. See Luckow andSummers, VIROLOGY 170:31 (1989). Other commercially available vectorsinclude, for example, PBlueBac4.5/V5-His; pBlueBacHis2; pMelBac;pBlueBac4.5 (Invitrogen Corp., Carlsbad, Calif.).

After insertion of the heterologous gene, the transfer vector and wildtype baculoviral genome are co-transfected into an insect cell host.Methods for introducing heterologous DNA into the desired site in thebaculovirus virus are known in the art. See SUMMERS AND SMITH, TEXASAGRICULTURAL EXPERIMENT STATION BULLETIN NO. 1555 (1987); Smith et al.,MOL. CELL. BIOL. (1983) 3:2156; Luckow and Summers, VIROLOGY (1989)170:31. For example, the insertion can be into a gene such as thepolyhedrin gene, by homologous double crossover recombination; insertioncan also be into a restriction enzyme site engineered into the desiredbaculovirus gene. See Miller et al., BIOESSAYS (1989) 11(4):91.

Transfection may be accomplished by electroporation. See TROTTER ANDWOOD, 39 METHODS IN MOLECULAR BIOLOGY (1995); Mann and King, J. GEN.VIROL. (1989) 70:3501. Alternatively, liposomes may be used to transfectthe insect cells with the recombinant expression vector and thebaculovirus. See, e.g., Liebman et al., BIOTECHNIQUES (1999) 26(1):36;Graves et al., BIOCHEMISTRY (1998) 37:6050; Nomura et al., J. BIOL.CHEM. (1998) 273(22):13570; Schmidt et al., PROTEIN EXPRESSION ANDPURIFICATION (1998) 12:323; Siffert et al., NATURE GENETICS (1998)18:45; TILKINS ET AL., CELL BIOLOGY: A LABORATORY HANDBOOK 145-154(1998); Cai et al., PROTEIN EXPRESSION AND PURIFICATION (1997) 10:263;Dolphin et al., NATURE GENETICS (1997) 17:491; Kost et al., GENE (1997)190:139; Jakobsson et al., J. BIOL. CHEM. (1996) 271:22203; Rowles etal., J. BIOL. CHEM. (1996) 271(37):22376; Reverey et al., J. BIOL. CHEM.(1996) 271(39):23607-10; Stanley et al., J. BIOL. CHEM. (1995) 270:4121;Sisk et al., J. V IROL. (1994) 68(2):766; and Peng et al., BIOTECHNIQUES (1993) 14(2):274. Commercially available liposomes include,for example, Cellfectin® and Lipofectin® (Invitrogen, Corp., Carlsbad,Calif.). In addition, calcium phosphate transfection may be used. SeeTROTTER AND WOOD, 39 METHODS IN MOLECULAR BIOLOGY (1995); Kitts, NAR(1990) 18(19):5667; and Mann and King, J. GEN. VIROL. (1989) 70:3501.

Baculovirus expression vectors usually contain a baculovirus promoter. Abaculovirus promoter is any DNA sequence capable of binding abaculovirus RNA polymerase and initiating the downstream (3′)transcription of a coding sequence (e.g., structural gene) into mRNA. Apromoter will have a transcription initiation region which is usuallyplaced proximal to the 5′ end of the coding sequence. This transcriptioninitiation region typically includes an RNA polymerase binding site anda transcription initiation site. A baculovirus promoter may also have asecond domain called an enhancer, which, if present, is usually distalto the structural gene. Moreover, expression may be either regulated orconstitutive.

Structural genes, abundantly transcribed at late times in the infectioncycle, provide particularly useful promoter sequences. Examples includesequences derived from the gene encoding the viral polyhedron protein(FRIESEN ET AL ., The Regulation of Baculovirus Gene Expression in THEMOLECULAR BIOLOGY OF BACULOVIRUSES (1986); EP 0 127 839 and 0 155 476)and the gene encoding the p10 protein (Vlak et al., J. GEN. VIROL.(1988) 69:765).

The newly formed baculovirus expression vector is packaged into aninfectious recombinant baculovirus and subsequently grown plaques may bepurified by techniques known to those of ordinary skill in the art. SeeMiller et al., BIOESSAYS (1989) 11(4):91; SUMMERS AND SMITH, TEXASAGRICULTURAL EXPERIMENT STATION BULLETIN NO. 1555 (1987).

Recombinant baculovirus expression vectors have been developed forinfection into several insect cells. For example, recombinantbaculoviruses have been developed for, inter alia, Aedes aegypti (ATCCNo. CCL-125), Bombyx mori (ATCC No. CRL-8910), Drosophila melanogaster(ATCC No. 1963), Spodoptera frugiperda, and Trichoplusia ni. See Wright,NATURE (1986) 321:718; Carbonell et al., J. VIROL. (1985) 56:153; Smithet al., MOL. CELL. BIOL. (1983) 3:2156. See generally, Fraser et al., INVITRO CELL. DEV. BIOL. (1989) 25:225. More specifically, the cell linesused for baculovirus expression vector systems commonly include, but arenot limited to, Sf9 (Spodoptera frugiperda) (ATCC No. CRL-1711), Sf21(Spodoptera frugiperda) (Invitrogen Corp., Cat. No. 11497-013 (Carlsbad,Calif.)), Tri-368 (Trichopulsia ni), and High-Five™ BTI-TN-5B1-4(Trichopulsia ni).

Cells and culture media are commercially available for both direct andfusion expression of heterologous polypeptides in abaculovirus/expression, and cell culture technology is generally knownto those of ordinary skill in the art.

E. Coli, Pseudomonas species, and other Prokaryotes Bacterial expressiontechniques are known to those of ordinary skill in the art. A widevariety of vectors are available for use in bacterial hosts. The vectorsmay be single copy or low or high multicopy vectors. Vectors may servefor cloning and/or expression. In view of the ample literatureconcerning vectors, commercial availability of many vectors, and evenmanuals describing vectors and their restriction maps andcharacteristics, no extensive discussion is required here. As iswell-known, the vectors normally involve markers allowing for selection,which markers may provide for cytotoxic agent resistance, prototrophy orimmunity. Frequently, a plurality of markers is present, which providefor different characteristics.

A bacterial promoter is any DNA sequence capable of binding bacterialRNA polymerase and initiating the downstream (3′) transcription of acoding sequence (e.g. structural gene) into mRNA. A promoter will have atranscription initiation region which is usually placed proximal to the5′ end of the coding sequence. This transcription initiation regiontypically includes an RNA polymerase binding site and a transcriptioninitiation site. A bacterial promoter may also have a second domaincalled an operator, that may overlap an adjacent RNA polymerase bindingsite at which RNA synthesis begins. The operator permits negativeregulated (inducible) transcription, as a gene repressor protein maybind the operator and thereby inhibit transcription of a specific gene.Constitutive expression may occur in the absence of negative regulatoryelements, such as the operator. In addition, positive regulation may beachieved by a gene activator protein binding sequence, which, if presentis usually proximal (5′) to the RNA polymerase binding sequence. Anexample of a gene activator protein is the catabolite activator protein(CAP), which helps initiate transcription of the lac operon inEscherichia coli (E. coli) [Raibaud et al., ANNU. REV. GENET. (1984)18:173]. Regulated expression may therefore be either positive ornegative, thereby either enhancing or reducing transcription.

Sequences encoding metabolic pathway enzymes provide particularly usefulpromoter sequences. Examples include promoter sequences derived fromsugar metabolizing enzymes, such as galactose, lactose (lac) [Chang etal., NATURE (1977) 198:1056], and maltose. Additional examples includepromoter sequences derived from biosynthetic enzymes such as tryptophan(trp) [Goeddel et al., NUC. ACIDS RES. (1980) 8:4057; Yelverton et al.,NUCL. ACIDS RES. (1981) 9:731; U.S. Pat. No. 4,738,921; EP Pub. Nos. 036776 and 121 775, which are incorporated by reference herein]. Theβ-galactosidase (bla) promoter system [Weissmann (1981) “The cloning ofinterferon and other mistakes.” In Interferon 3 (Ed. I. Gresser)],bacteriophage lambda PL [Shimatake et al., NATURE (1981) 292:128] and T5[U.S. Pat. No. 4,689,406, which are incorporated by reference herein]promoter systems also provide useful promoter sequences. Preferredmethods of the present invention utilize strong promoters, such as theT7 promoter to induce GH, e.g., hGH polypeptides at high levels.Examples of such vectors are known to those of ordinary skill in the artand include the pET29 series from Novagen, and the pPOP vectorsdescribed in WO99/05297, which is incorporated by reference herein. Suchexpression systems produce high levels of GH, e.g., hGH polypeptides inthe host without compromising host cell viability or growth parameters.pET19 (Novagen) is another vector known in the art.

In addition, synthetic promoters which do not occur in nature alsofunction as bacterial promoters. For example, transcription activationsequences of one bacterial or bacteriophage promoter may be joined withthe operon sequences of another bacterial or bacteriophage promoter,creating a synthetic hybrid promoter [U.S. Pat. No. 4,551,433, which isincorporated by reference herein]. For example, the tac promoter is ahybrid trp-lac promoter comprised of both trp promoter and lac operonsequences that is regulated by the lac repressor [Amann et al., GENE(1983) 25:167; de Boer et al., PROC. NATL. ACAD. SCI. (1983) 80:21].Furthermore, a bacterial promoter can include naturally occurringpromoters of non-bacterial origin that have the ability to bindbacterial RNA polymerase and initiate transcription. A naturallyoccurring promoter of non-bacterial origin can also be coupled with acompatible RNA polymerase to produce high levels of expression of somegenes in prokaryotes. The bacteriophage T7 RNA polymerase/promotersystem is an example of a coupled promoter system [Studier et al., J.MOL. BIOL. (1986) 189:113; Tabor et al., Proc Natl. Acad. Sci. (1985)82:1074]. In addition, a hybrid promoter can also be comprised of abacteriophage promoter and an E. coli operator region (EP Pub. No. 267851).

In addition to a functioning promoter sequence, an efficient ribosomebinding site is also useful for the expression of foreign genes inprokaryotes. In E. coli, the ribosome binding site is called theShine-Dalgarno (SD) sequence and includes an initiation codon (ATG) anda sequence 3-9 nucleotides in length located 3-11 nucleotides upstreamof the initiation codon [Shine et al., NATURE (1975) 254:34]. The SDsequence is thought to promote binding of mRNA to the ribosome by thepairing of bases between the SD sequence and the 3′ and of E. coli 16SrRNA [Steitz et al. “Genetic signals and nucleotide sequences inmessenger RNA”, In Biological Regulation and Development: GeneExpression (Ed. R. F. Goldberger, 1979)]. To express eukaryotic genesand prokaryotic genes with weak ribosome-binding site [Sambrook et al.“Expression of cloned genes in Escherichia coli”, Molecular Cloning: ALaboratory Manual, 1989].

The term “bacterial host” or “bacterial host cell” refers to a bacterialthat can be, or has been, used as a recipient for recombinant vectors orother transfer DNA. The term includes the progeny of the originalbacterial host cell that has been transfected. It is understood that theprogeny of a single parental cell may not necessarily be completelyidentical in morphology or in genomic or total DNA complement to theoriginal parent, due to accidental or deliberate mutation. Progeny ofthe parental cell that are sufficiently similar to the parent to becharacterized by the relevant property, such as the presence of anucleotide sequence encoding a GH, e.g., hGH polypeptide, are includedin the progeny intended by this definition.

The selection of suitable host bacteria for expression of GH, e.g., hGHpolypeptides is known to those of ordinary skill in the art. Inselecting bacterial hosts for expression, suitable hosts may includethose shown to have, inter alia, good inclusion body formation capacity,low proteolytic activity, and overall robustness. Bacterial hosts aregenerally available from a variety of sources including, but not limitedto, the Bacterial Genetic Stock Center, Department of Biophysics andMedical Physics, University of California (Berkeley, Calif.); and theAmerican Type Culture Collection (“ATCC”) (Manassas, Va.).Industrial/pharmaceutical fermentation generally use bacterial derivedfrom K strains (e.g. W3110) or from bacteria derived from B strains(e.g. BL21). These strains are particularly useful because their growthparameters are extremely well known and robust. In addition, thesestrains are non-pathogenic, which is commercially important for safetyand environmental reasons. Other examples of suitable E. coli hostsinclude, but are not limited to, strains of BL21, DH10B, or derivativesthereof. In another embodiment of the methods of the present invention,the E. coli host is a protease minus strain including, but not limitedto, OMP- and LON-. The host cell strain may be a species of Pseudomonas,including but not limited to, Pseudomonas fluorescens, Pseudomonasaeruginosa, and Pseudomonas putida. Pseudomonas fluorescens biovar 1,designated strain MB101, is known to be useful for recombinantproduction and is available for therapeutic protein productionprocesses. Examples of a Pseudomonas expression system include thesystem available from The Dow Chemical Company as a host strain(Midland, Mich. available on the World Wide Web at dow.com). U.S. Pat.Nos. 4,755,465 and 4,859,600, which are incorporated by referenceherein, describe the use of Pseudomonas strains as a host cell for GH,e.g., hGH production.

Once a recombinant host cell strain has been established (i.e., theexpression construct has been introduced into the host cell and hostcells with the proper expression construct are isolated), therecombinant host cell strain is cultured under conditions appropriatefor production of GH, e.g., hGH polypeptides. As will be apparent to oneof skill in the art, the method of culture of the recombinant host cellstrain will be dependent on the nature of the expression constructutilized and the identity of the host cell. Recombinant host strains arenormally cultured using methods that are known to those of ordinaryskill in the art. Recombinant host cells are typically cultured inliquid medium containing assimilatable sources of carbon, nitrogen, andinorganic salts and, optionally, containing vitamins, amino acids,growth factors, and other proteinaceous culture supplements known tothose of ordinary skill in the art. Liquid media for culture of hostcells may optionally contain antibiotics or anti-fungals to prevent thegrowth of undesirable microorganisms and/or compounds including, but notlimited to, antibiotics to select for host cells containing theexpression vector.

Recombinant host cells may be cultured in batch or continuous formats,with either cell harvesting (in the case where the GH, e.g., hGHpolypeptide accumulates intracellularly) or harvesting of culturesupernatant in either batch or continuous formats. For production inprokaryotic host cells, batch culture and cell harvest are preferred.

The GH, e.g., hGH polypeptides of the present invention are normallypurified after expression in recombinant systems. The GH, e.g., hGHpolypeptide may be purified from host cells or culture medium by avariety of methods known to the art. GH, e.g., hGH polypeptides producedin bacterial host cells may be poorly soluble or insoluble (in the formof inclusion bodies). In one embodiment of the present invention, aminoacid substitutions may readily be made in the GH, e.g., hGH polypeptidethat are selected for the purpose of increasing the solubility of therecombinantly produced protein utilizing the methods disclosed herein aswell as those known in the art. In the case of insoluble protein, theprotein may be collected from host cell lysates by centrifugation andmay further be followed by homogenization of the cells. In the case ofpoorly soluble protein, compounds including, but not limited to,polyethylene imine (PEI) may be added to induce the precipitation ofpartially soluble protein. The precipitated protein may then beconveniently collected by centrifugation. Recombinant host cells may bedisrupted or homogenized to release the inclusion bodies from within thecells using a variety of methods known to those of ordinary skill in theart. Host cell disruption or homogenization may be performed using wellknown techniques including, but not limited to, enzymatic celldisruption, sonication, dounce homogenization, or high pressure releasedisruption. In one embodiment of the method of the present invention,the high pressure release technique is used to disrupt the E. coli hostcells to release the inclusion bodies of the GH, e.g., hGH polypeptides.When handling inclusion bodies of GH, e.g., hGH polypeptide, it may beadvantageous to minimize the homogenization time on repetitions in orderto maximize the yield of inclusion bodies without loss due to factorssuch as solubilization, mechanical shearing or proteolysis.

Insoluble or precipitated GH, e.g., hGH polypeptide may then besolubilized using any of a number of suitable solubilization agentsknown to the art. The GH, e.g., hGH polypeptide may be solubilized withurea or guanidine hydrochloride. The volume of the solubilized GH, e.g.,hGH polypeptide should be minimized so that large batches may beproduced using conveniently manageable batch sizes. This factor may besignificant in a large-scale commercial setting where the recombinanthost may be grown in batches that are thousands of liters in volume. Inaddition, when manufacturing GH, e.g., hGH polypeptide in a large-scalecommercial setting, in particular for human pharmaceutical uses, theavoidance of harsh chemicals that can damage the machinery andcontainer, or the protein product itself, should be avoided, ifpossible. It has been shown in the method of the present invention thatthe milder denaturing agent urea can be used to solubilize the GH, e.g.,hGH polypeptide inclusion bodies in place of the harsher denaturingagent guanidine hydrochloride. The use of urea significantly reduces therisk of damage to stainless steel equipment utilized in themanufacturing and purification process of GH, e.g., hGH polypeptidewhile efficiently solubilizing the GH, e.g., hGH polypeptide inclusionbodies.

In the case of soluble hGH protein, the hGH may be secreted into theperiplasmic space or into the culture medium. In addition, soluble hGHmay be present in the cytoplasm of the host cells. It may be desired toconcentrate soluble hGH prior to performing purification steps. Standardtechniques known to those of ordinary skill in the art may be used toconcentrate soluble hGH from, for example, cell lysates or culturemedium. In addition, standard techniques known to those of ordinaryskill in the art may be used to disrupt host cells and release solublehGH from the cytoplasm or periplasmic space of the host cells.

When GH, e.g., hGH polypeptide is produced as a fusion protein, thefusion sequence may be removed. Removal of a fusion sequence may beaccomplished by enzymatic or chemical cleavage. Enzymatic removal offusion sequences may be accomplished using methods known to those ofordinary skill in the art. The choice of enzyme for removal of thefusion sequence will be determined by the identity of the fusion, andthe reaction conditions will be specified by the choice of enzyme aswill be apparent to one of ordinary skill in the art. Chemical cleavagemay be accomplished using reagents known to those of ordinary skill inthe art, including but not limited to, cyanogen bromide, TEV protease,and other reagents. The cleaved GH, e.g., hGH polypeptide may bepurified from the cleaved fusion sequence by methods known to those ofordinary skill in the art. Such methods will be determined by theidentity and properties of the fusion sequence and the GH, e.g., hGHpolypeptide, as will be apparent to one of ordinary skill in the art.Methods for purification may include, but are not limited to,size-exclusion chromatography, hydrophobic interaction chromatography,ion-exchange chromatography or dialysis or any combination thereof.

The GH, e.g., hGH polypeptide may also be purified to remove DNA fromthe protein solution. DNA may be removed by any suitable method known tothe art, such as precipitation or ion exchange chromatography, but maybe removed by precipitation with a nucleic acid precipitating agent,such as, but not limited to, protamine sulfate. The GH, e.g., hGHpolypeptide may be separated from the precipitated DNA using standardwell known methods including, but not limited to, centrifugation orfiltration. Removal of host nucleic acid molecules is an importantfactor in a setting where the GH, e.g., hGH polypeptide is to be used totreat humans and the methods of the present invention reduce host cellDNA to pharmaceutically acceptable levels.

Methods for small-scale or large-scale fermentation can also be used inprotein expression, including but not limited to, fermentors, shakeflasks, fluidized bed bioreactors, hollow fiber bioreactors, rollerbottle culture systems, and stirred tank bioreactor systems. Each ofthese methods can be performed in a batch, fed-batch, or continuous modeprocess.

Human GH polypeptides of the invention can generally be recovered usingmethods standard in the art. For example, culture medium or cell lysatecan be centrifuged or filtered to remove cellular debris. Thesupernatant may be concentrated or diluted to a desired volume ordiafiltered into a suitable buffer to condition the preparation forfurther purification. Further purification of the GH, e.g., hGHpolypeptide of the present invention includes separating deamidated andclipped forms of the GH, e.g., hGH polypeptide variant from the intactform.

Any of the following exemplary procedures can be employed forpurification of GH, e.g., hGH polypeptides of the invention: affinitychromatography; anion- or cation-exchange chromatography (using,including but not limited to, DEAE SEPHAROSE); chromatography on silica;reverse phase HPLC; gel filtration (using, including but not limited to,SEPHADEX G-75); hydrophobic interaction chromatography; size-exclusionchromatography, metal-chelate chromatography;ultrafiltration/diafiltration; ethanol precipitation; ammonium sulfateprecipitation; chromatofocusing; displacement chromatography;electrophoretic procedures (including but not limited to preparativeisoelectric focusing), differential solubility (including but notlimited to ammonium sulfate precipitation), SDS-PAGE, or extraction.

Proteins of the present invention, including but not limited to,proteins comprising unnatural amino acids, peptides comprising unnaturalamino acids, antibodies to proteins comprising unnatural amino acids,binding partners for proteins comprising unnatural amino acids, etc.,can be purified, either partially or substantially to homogeneity,according to standard procedures known to and used by those of skill inthe art. Accordingly, polypeptides of the invention can be recovered andpurified by any of a number of methods known to those of ordinary skillin the art, including but not limited to, ammonium sulfate or ethanolprecipitation, acid or base extraction, column chromatography, affinitycolumn chromatography, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,hydroxylapatite chromatography, lectin chromatography, gelelectrophoresis and the like. Protein refolding steps can be used, asdesired, in making correctly folded mature proteins. High performanceliquid chromatography (HPLC), affinity chromatography or other suitablemethods can be employed in final purification steps where high purity isdesired. In one embodiment, antibodies made against unnatural aminoacids (or proteins or peptides comprising unnatural amino acids) areused as purification reagents, including but not limited to, foraffinity-based purification of proteins or peptides comprising one ormore unnatural amino acid(s). Once purified, partially or tohomogeneity, as desired, the polypeptides are optionally used for a widevariety of utilities, including but not limited to, as assay components,therapeutics, prophylaxis, diagnostics, research reagents, and/or asimmunogens for antibody production.

In addition to other references noted herein, a variety ofpurification/protein folding methods are known to those of ordinaryskill in the art, including, but not limited to, those set forth in R.Scopes, Protein Purification, Springer-Verlag, N.Y. (1982); Deutscher,Methods in Enzymology Vol. 182: Guide to Protein Purification, AcademicPress, Inc. N.Y. (1990); Sandana, (1997) Bioseparation of Proteins,Academic Press, Inc.; Bollag et al. (1996) Protein Methods, 2nd EditionWiley-Liss, NY; Walker, (1996) The Protein Protocols Handbook HumanaPress, NJ, Harris and Angal, (1990) Protein Purification Applications: APractical Approach IRL Press at Oxford, Oxford, England; Harris andAngal, Protein Purification Methods: A Practical Approach IRL Press atOxford, Oxford, England; Scopes, (1993) Protein Purification: Principlesand Practice 3rd Edition Springer Verlag, NY; Janson and Ryden, (1998)Protein Purification: Principles, High Resolution Methods andApplications, Second Edition Wiley-VCH, NY; and Walker (1998), ProteinProtocols on CD-ROM Humana Press, NJ; and the references cited therein.

One advantage of producing a protein or polypeptide of interest with anunnatural amino acid in a eukaryotic host cell or non-eukaryotic hostcell is that typically the proteins or polypeptides will be folded intheir native conformations. However, in certain embodiments of theinvention, those of skill in the art will recognize that, aftersynthesis, expression and/or purification, proteins or peptides canpossess a conformation different from the desired conformations of therelevant polypeptides. In one aspect of the invention, the expressedprotein is optionally denatured and then renatured. This is accomplishedutilizing methods known in the art, including but not limited to, byadding a chaperonin to the protein or polypeptide of interest, bysolubilizing the proteins in a chaotropic agent such as guanidine HCl,utilizing protein disulfide isomerase, etc.

In general, it is occasionally desirable to denature and reduceexpressed polypeptides and then to cause the polypeptides to re-foldinto the preferred conformation. For example, guanidine, urea, DTT, DTE,and/or a chaperonin can be added to a translation product of interest.Methods of reducing, denaturing and renaturing proteins are known tothose of ordinary skill in the art (see, the references above, andDebinski, et al. (1993) J. Biol. Chem., 268: 14065-14070; Kreitman andPastan (1993) Bioconjug. Chem., 4: 581-585; and Buchner, et al., (1992)Anal. Biochem., 205: 263-270). Debinski, et al., for example, describethe denaturation and reduction of inclusion body proteins inguanidine-DTE. The proteins can be refolded in a redox buffercontaining, including but not limited to, oxidized glutathione andL-arginine. Refolding reagents can be flowed or otherwise moved intocontact with the one or more polypeptide or other expression product, orvice-versa.

In the case of prokaryotic production of GH, e.g., hGH polypeptide, theGH, e.g., hGH polypeptide thus produced may be misfolded and thus lacksor has reduced biological activity. The bioactivity of the protein maybe restored by “refolding”. In general, misfolded GH, e.g., hGHpolypeptide is refolded by solubilizing (where the GH, e.g., hGHpolypeptide is also insoluble), unfolding and reducing the polypeptidechain using, for example, one or more chaotropic agents (e.g. ureaand/or guanidine) and a reducing agent capable of reducing disulfidebonds (e.g. dithiothreitol, DTT or 2-mercaptoethanol, 2-ME). At amoderate concentration of chaotrope, an oxidizing agent is then added(e.g., oxygen, cystine or cystamine), which allows the reformation ofdisulfide bonds. GH, e.g., hGH polypeptide may be refolded usingstandard methods known in the art, such as those described in U.S. Pat.Nos. 4,511,502, 4,511,503, and 4,512,922, which are incorporated byreference herein. The GH, e.g., hGH polypeptide may also be cofoldedwith other proteins to form heterodimers or heteromultimers.

After refolding or cofolding, the GH, e.g. hGH polypeptide may befurther purified. Purification of GH, e.g., hGH may be accomplishedusing a variety of techniques known to those of ordinary skill in theart, including hydrophobic interaction chromatography, size exclusionchromatography, ion exchange chromatography, reverse-phase highperformance liquid chromatography, affinity chromatography, and the likeor any combination thereof. Additional purification may also include astep of drying or precipitation of the purified protein.

After purification, GH, e.g., hGH may be exchanged into differentbuffers and/or concentrated by any of a variety of methods known to theart, including, but not limited to, diafiltration and dialysis. GH,e.g., hGH that is provided as a single purified protein may be subjectto aggregation and precipitation.

The purified GH, e.g., hGH may be at least 90% pure (as measured byreverse phase high performance liquid chromatography, RP-HPLC, or sodiumdodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE) or atleast 95% pure, or at least 98% pure, or at least 99% or greater pure.Regardless of the exact numerical value of the purity of the GH, e.g.,hGH, the GH, e.g., hGH is may be sufficiently pure for use as apharmaceutical product or for further processing, such as conjugationwith a water soluble polymer such as PEG.

Certain GH, e.g., hGH molecules may be used as therapeutic agents in theabsence of other active ingredients or proteins (other than excipients,carriers, and stabilizers, serum albumin and the like), or they may becomplexed with another protein or a polymer.

General Purification Methods Any one of a variety of isolation steps maybe performed on the cell lysate, extract, culture medium, inclusionbodies, periplasmic space of the host cells, cytoplasm of the hostcells, or other material, comprising GH, e.g., hGH polypeptide or on anyGH, e.g., hGH polypeptide mixtures resulting from any isolation stepsincluding, but not limited to, affinity chromatography, ion exchangechromatography, hydrophobic interaction chromatography, gel filtrationchromatography, high performance liquid chromatography (“HPLC”),reversed phase-HPLC (“RP-HPLC”), expanded bed adsorption, or anycombination and/or repetition thereof and in any appropriate order.

Equipment and other necessary materials used in performing thetechniques described herein are commercially available. Pumps, fractioncollectors, monitors, recorders, and entire systems are available from,for example, Applied Biosystems (Foster City, Calif.), Bio-RadLaboratories, Inc. (Hercules, Calif.), and Amersham Biosciences, Inc.(Piscataway, N.J.). Chromatographic materials including, but not limitedto, exchange matrix materials, media, and buffers are also availablefrom such companies.

Equilibration, and other steps in the column chromatography processesdescribed herein such as washing and elution, may be more rapidlyaccomplished using specialized equipment such as a pump. Commerciallyavailable pumps include, but are not limited to, HILOAD® Pump P-50,Peristaltic Pump P-1, Pump P-901, and Pump P-903 (Amersham Biosciences,Piscataway, N.J.).

Examples of fraction collectors include RediFrac Fraction Collector,FRAC-100 and FRAC-200 Fraction Collectors, and SUPERFRAC® FractionCollector (Amersham Biosciences, Piscataway, N.J.). Mixers are alsoavailable to form pH and linear concentration gradients. Commerciallyavailable mixers include Gradient Mixer GM-1 and In-Line Mixers(Amersham Biosciences, Piscataway, N.J.).

The chromatographic process may be monitored using any commerciallyavailable monitor. Such monitors may be used to gather information likeUV, pH, and conductivity. Examples of detectors include Monitor UV-1,UVICORD® S II, Monitor UV-M II, Monitor UV-900, Monitor UPC-900, MonitorpH/C-900, and Conductivity Monitor (Amersham Biosciences, Piscataway,N.J.). Indeed, entire systems are commercially available including thevarious AKTA® systems from Amersham Biosciences (Piscataway, N.J.).

In one embodiment of the present invention, for example, the GH, e.g.,hGH polypeptide may be reduced and denatured by first denaturing theresultant purified GH, e.g., hGH polypeptide in urea, followed bydilution into TRIS buffer containing a reducing agent (such as DTT) at asuitable pH. In another embodiment, the GH, e.g., hGH polypeptide isdenatured in urea in a concentration range of between about 2 M to about9 M, followed by dilution in TRIS buffer at a pH in the range of about5.0 to about 8.0. The refolding mixture of this embodiment may then beincubated. In one embodiment, the refolding mixture is incubated at roomtemperature for four to twenty-four hours. The reduced and denatured GH,e.g., hGH polypeptide mixture may then be further isolated or purified.

As stated herein, the pH of the first GH, e.g., hGH polypeptide mixturemay be adjusted prior to performing any subsequent isolation steps. Inaddition, the first GH, e.g., hGH polypeptide mixture or any subsequentmixture thereof may be concentrated using techniques known in the art.Moreover, the elution buffer comprising the first GH, e.g., hGHpolypeptide mixture or any subsequent mixture thereof may be exchangedfor a buffer suitable for the next isolation step using techniques knownto those of ordinary skill in the art.

Ion Exchange Chromatography In one embodiment, and as an optional,additional step, ion exchange chromatography may be performed on thefirst GH, e.g., hGH polypeptide mixture. See generally ION EXCHANGECHROMATOGRAPHY: PRINCIPLES AND METHODS (Cat. No. 18-1114-21, AmershamBiosciences (Piscataway, N.J.)). Commercially available ion exchangecolumns include HITRAP®, HIPREP®, and HILOAD® Columns (AmershamBiosciences, Piscataway, N.J.). Such columns utilize strong anionexchangers such as Q SEPHAROSE® Fast Flow, Q SEPHAROSE® HighPerformance, and Q SEPHAROSE® XL; strong cation exchangers such as SPSEPHAROSE® High Performance, SP SEPHAROSE® Fast Flow, and SP SEPHAROSE®XL; weak anion exchangers such as DEAE SEPHAROSE® Fast Flow; and weakcation exchangers such as CM SEPHAROSE® Fast Flow (Amersham Biosciences,Piscataway, N.J.). Anion or cation exchange column chromatography may beperformed on the GH, e.g., hGH-polypeptide at any stage of thepurification process to isolate substantially purified GH, e.g., hGHpolypeptide. The cation exchange chromatography step may be performedusing any suitable cation exchange matrix. Useful cation exchangematrices include, but are not limited to, fibrous, porous, non-porous,microgranular, beaded, or cross-linked cation exchange matrix materials.Such cation exchange matrix materials include, but are not limited to,cellulose, agarose, dextran, polyacrylate, polyvinyl, polystyrene,silica, polyether, or composites of any of the foregoing.

The cation exchange matrix may be any suitable cation exchangerincluding strong and weak cation exchangers. Strong cation exchangersmay remain ionized over a wide pH range and thus, may be capable ofbinding GH, e.g., hGH over a wide pH range. Weak cation exchangers,however, may lose ionization as a function of pH. For example, a weakcation exchanger may lose charge when the pH drops below about pH 4 orpH 5. Suitable strong cation exchangers include, but are not limited to,charged functional groups such as sulfopropyl (SP), methyl sulfonate(S), or sulfoethyl (SE). The cation exchange matrix may be a strongcation exchanger, preferably having a GH, e.g., hGH binding pH range ofabout 2.5 to about 6.0. Alternatively, the strong cation exchanger mayhave a GH, e.g., hGH binding pH range of about pH 2.5 to about pH 5.5.The cation exchange matrix may be a strong cation exchanger having a GH,e.g., hGH binding pH of about 3.0. Alternatively, the cation exchangematrix may be a strong cation exchanger, preferably having a GH, e.g.,hGH binding pH range of about 6.0 to about 8.0. The cation exchangematrix may be a strong cation exchanger preferably having a GH, e.g.,hGH binding pH range of about 8.0 to about 12.5. Alternatively, thestrong cation exchanger may have a GH, e.g., hGH binding pH range ofabout pH 8.0 to about pH 12.0.

Prior to loading the GH, e.g., hGH, the cation exchange matrix may beequilibrated, for example, using several column volumes of a dilute,weak acid, e.g., four column volumes of 20 mM acetic acid, pH 3.Following equilibration, the GH, e.g., hGH may be added and the columnmay be washed one to several times, prior to elution of substantiallypurified GH, e.g., hGH, also using a weak acid solution such as a weakacetic acid or phosphoric acid solution. For example, approximately 2-4column volumes of 20 mM acetic acid, pH 3, may be used to wash thecolumn. Additional washes using, e.g., 2-4 column volumes of 0.05 Msodium acetate, pH 5.5, or 0.05 M sodium acetate mixed with 0.1 M sodiumchloride, pH 5.5, may also be used. Alternatively, using methods knownin the art, the cation exchange matrix may be equilibrated using severalcolumn volumes of a dilute, weak base.

Alternatively, substantially purified GH, e.g., hGH may be eluted bycontacting the cation exchanger matrix with a buffer having asufficiently low pH or ionic strength to displace the GH, e.g., hGH fromthe matrix. The pH of the elution buffer may range from about pH 2.5 toabout pH 6.0. More specifically, the pH of the elution buffer may rangefrom about pH 2.5 to about pH 5.5, about pH 2.5 to about pH 5.0. Theelution buffer may have a pH of about 3.0. In addition, the quantity ofelution buffer may vary widely and will generally be in the range ofabout 2 to about 10 column volumes.

Following adsorption of the GH, e.g., hGH polypeptide to the cationexchanger matrix, substantially purified hGH polypeptide may be elutedby contacting the matrix with a buffer having a sufficiently high pH orionic strength to displace the GH, e.g., hGH polypeptide from thematrix. Suitable buffers for use in high pH elution of substantiallypurified GH, e.g., hGH polypeptide may find use herein include, but arenot limited to, citrate, phosphate, formate, acetate, HEPES, and MESbuffers ranging in concentration from at least about 5 mM to at leastabout 100 mM.

Reverse-Phase Chromatography RP-HPLC may be performed to purify proteinsfollowing suitable protocols that are known to those of ordinary skillin the art. See, e.g., Pearson et al., ANAL BIOCHEM. (1982) 124:217-230(1982); Rivier et al., J. CHROM. (1983) 268:112-119; Kunitani et al., J.CHROM. (1986) 359:391-402. RP-HPLC may be performed on the GH, e.g., hGHpolypeptide to isolate substantially purified GH, e.g., hGH polypeptide.In this regard, silica derivatized resins with alkyl functionalitieswith a wide variety of lengths, including, but not limited to, at leastabout C₃ to at least about C₃₀, at least about C₃ to at least about C₂₀,or at least about C₃ to at least about C₁₈, resins may be used.Alternatively, a polymeric resin may be used. For example, TosoHaasAmberchrome CG1000sd resin may be used, which is a styrene polymerresin. Cyano or polymeric resins with a wide variety of alkyl chainlengths may also be used. Furthermore, the RP-HPLC column may be washedwith a solvent such as ethanol. The Source RP column is another exampleof a RP-HPLC column.

A suitable elution buffer containing an ion pairing agent and an organicmodifier such as methanol, isopropanol, tetrahydrofuran, acetonitrile orethanol, may be used to elute the GH, e.g., hGH polypeptide from theRP-HPLC column. The most commonly used ion pairing agents include, butare not limited to, acetic acid, formic acid, perchloric acid,phosphoric acid, trifluoroacetic acid, heptafluorobutyric acid,triethylamine, tetramethylammonium, tetrabutylammonium, andtriethylammonium acetate. Elution may be performed using one or moregradients or isocratic conditions, with gradient conditions preferred toreduce the separation time and to decrease peak width. Another methodinvolves the use of two gradients with different solvent concentrationranges. Examples of suitable elution buffers for use herein may include,but are not limited to, ammonium acetate and acetonitrile solutions.

Hydrophobic Interaction Chromatography Purification TechniquesHydrophobic interaction chromatography (HIC) may be performed on the GH,e.g., hGH polypeptide. See generally HYDROPHOBIC INTERACTIONCHROMATOGRAPHY HANDBOOK: PRINCIPLES AND METHODS (Cat. No. 18-1020-90,Amersham Biosciences (Piscataway, N.J.) which is incorporated byreference herein. Suitable HIC matrices may include, but are not limitedto, alkyl- or aryl-substituted matrices, such as butyl-, hexyl-, octyl-or phenyl-substituted matrices including agarose, cross-linked agarose,sepharose, cellulose, silica, dextran, polystyrene, poly(methacrylate)matrices, and mixed mode resins, including but not limited to, apolyethyleneamine resin or a butyl- or phenyl-substitutedpoly(methacrylate) matrix. Commercially available sources forhydrophobic interaction column chromatography include, but are notlimited to, HITRAP®, HIPREP®, and HILOAD® columns (Amersham Biosciences,Piscataway, N.J.). Briefly, prior to loading, the HIC column may beequilibrated using standard buffers known to those of ordinary skill inthe art, such as an acetic acid/sodium chloride solution or HEPEScontaining ammonium sulfate. Ammonium sulfate may be used as the bufferfor loading the HIC column. After loading the GH, e.g., hGH polypeptide,the column may then washed using standard buffers and conditions toremove unwanted materials but retaining the GH, e.g., hGH polypeptide onthe HIC column. The GH, e.g., hGH polypeptide may be eluted with about 3to about 10 column volumes of a standard buffer, such as a HEPES buffercontaining EDTA and lower ammonium sulfate concentration than theequilibrating buffer, or an acetic acid/sodium chloride buffer, amongothers. A decreasing linear salt gradient using, for example, a gradientof potassium phosphate, may also be used to elute the GH, e.g., hGHmolecules. The eluant may then be concentrated, for example, byfiltration such as diafiltration or ultrafiltration. Diafiltration maybe utilized to remove the salt used to elute the GH, e.g., hGHpolypeptide.

Other Purification Techniques Yet another isolation step using, forexample, gel filtration (GEL FILTRATION: PRINCIPLES AND METHODS (Cat.No. 18-1022-18, Amersham Biosciences, Piscataway, N.J.) which isincorporated by reference herein, hydroxyapatite chromatography(suitable matrices include, but are not limited to, HA-Ultrogel, HighResolution (Calbiochem), CHT Ceramic Hydroxyapatite (BioRad), Bio-GelHTP Hydroxyapatite (BioRad)), HPLC, expanded bed adsorption,ultrafiltration, diafiltration, lyophilization, and the like, may beperformed on the first GH, e.g., hGH polypeptide mixture or anysubsequent mixture thereof, to remove any excess salts and to replacethe buffer with a suitable buffer for the next isolation step or evenformulation of the final drug product.

The yield of GH, e.g., hGH polypeptide, including substantially purifiedGH, e.g., hGH polypeptide, may be monitored at each step describedherein using techniques known to those of ordinary skill in the art.Such techniques may also be used to assess the yield of substantiallypurified GH, e.g., hGH polypeptide following the last isolation step.For example, the yield of GH, e.g., hGH polypeptide may be monitoredusing any of several reverse phase high pressure liquid chromatographycolumns, having a variety of alkyl chain lengths such as cyano RP-HPLC,C₁₈RP-HPLC; as well as cation exchange HPLC and gel filtration HPLC.

In specific embodiments of the present invention, the yield of GH, e.g.,hGH after each purification step may be at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 55%, at least about 60%, at least about 65%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 91%, at least about 92%, at leastabout 93%, at least about 94%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, at least about 99%, at leastabout 99.9%, or at least about 99.99%, of the GH, e.g., hGH in thestarting material for each purification step.

Purity may be determined using standard techniques, such as SDS-PAGE, orby measuring GH, e.g., hGH polypeptide using Western blot and ELISAassays. For example, polyclonal antibodies may be generated againstproteins isolated from negative control yeast fermentation and thecation exchange recovery. The antibodies may also be used to probe forthe presence of contaminating host cell proteins.

RP-HPLC material Vydac C4 (Vydac) consists of silica gel particles, thesurfaces of which carry C4-alkyl chains. The separation of GH, e.g., hGHpolypeptide from the proteinaceous impurities is based on differences inthe strength of hydrophobic interactions. Elution is performed with anacetonitrile gradient in diluted trifluoroacetic acid. Preparative HPLCis performed using a stainless steel column (filled with 2.8 to 3.2liter of Vydac C4 silicagel). The Hydroxyapatite Ultrogel eluate isacidified by adding trifluoroacetic acid and loaded onto the Vydac C4column. For washing and elution an acetonitrile gradient in dilutedtrifluoroacetic acid is used. Fractions are collected and immediatelyneutralized with phosphate buffer. The GH, e.g., hGH polypeptidefractions which are within the IPC limits are pooled.

DEAE Sepharose (Pharmacia) material consists of diethylaminoethyl(DEAE)-groups which are covalently bound to the surface of Sepharosebeads. The binding of GH, e.g., hGH polypeptide to the DEAE groups ismediated by ionic interactions. Acetonitrile and trifluoroacetic acidpass through the column without being retained. After these substanceshave been washed off, trace impurities are removed by washing the columnwith acetate buffer at a low pH. Then the column is washed with neutralphosphate buffer and GH, e.g., hGH polypeptide is eluted with a bufferwith increased ionic strength. The column is packed with DEAE Sepharosefast flow. The column volume is adjusted to assure a GH, e.g., hGHpolypeptide load in the range of 3-10 mg GH, e.g., hGH polypeptide/mlgel. The column is washed with water and equilibration buffer(sodium/potassium phosphate). The pooled fractions of the HPLC eluateare loaded and the column is washed with equilibration buffer. Then thecolumn is washed with washing buffer (sodium acetate buffer) followed bywashing with equilibration buffer. Subsequently, GH, e.g., hGHpolypeptide is eluted from the column with elution buffer (sodiumchloride, sodium/potassium phosphate) and collected in a single fractionin accordance with the master elution profile. The eluate of the DEAESepharose column is adjusted to the specified conductivity. Theresulting drug substance is sterile filtered into Teflon bottles andstored at −70° C.

Additional methods that may be employed include, but are not limited to,steps to remove endotoxins. Endotoxins are lipopoly-saccharides (LPSs)which are located on the outer membrane of Gram-negative host cells,such as, for example, Escherichia coli. Methods for reducing endotoxinlevels are known to one of ordinary skill in the art and include, butare not limited to, purification techniques using silica supports, glasspowder or hydroxyapatite, reverse-phase, affinity, size-exclusion,anion-exchange chromatography, hydrophobic interaction chromatography, acombination of these methods, and the like. Modifications or additionalmethods may be required to remove contaminants such as co-migratingproteins from the polypeptide of interest. Methods for measuringendotoxin levels are known to one of ordinary skill in the art andinclude, but are not limited to, Limulus Amebocyte Lysate (LAL) assays.

A wide variety of methods and procedures can be used to assess the yieldand purity of a GH, e.g., hGH protein one or more non-naturally encodedamino acids, including but not limited to, the Bradford assay, SDS-PAGE,silver stained SDS-PAGE, coomassie stained SDS-PAGE, mass spectrometry(including but not limited to, MALDI-TOF) and other methods forcharacterizing proteins known to one of ordinary skill in the art.

Additional methods include, but are not limited to: SDS-PAGE coupledwith protein staining methods, immunoblotting, matrix assisted laserdesorption/ionization-mass spectrometry (MALDI-MS), liquidchromatography/mass spectrometry, isoelectric focusing, analytical anionexchange, chromatofocusing, and circular dichroism.

VIII. Expression in Alternate Systems

Several strategies have been employed to introduce unnatural amino acidsinto proteins in non-recombinant host cells, mutagenized host cells, orin cell-free systems. These systems are also suitable for use in makingthe GH, e.g., hGH polypeptides of the present invention. Derivatizationof amino acids with reactive side-chains such as Lys, Cys and Tyrresulted in the conversion of lysine to N²-acetyl-lysine. Chemicalsynthesis also provides a straightforward method to incorporateunnatural amino acids. With the recent development of enzymatic ligationand native chemical ligation of peptide fragments, it is possible tomake larger proteins. See, e.g., P. E. Dawson and S. B. H. Kent, Annu.Rev. Biochem, 69:923 (2000). Chemical peptide ligation and nativechemical ligation are described in U.S. Pat. No. 6,184,344, U.S. PatentPublication No. 2004/0138412, U.S. Patent Publication No. 2003/0208046,WO 02/098902, and WO 03/042235, which are incorporated by referenceherein. A general in vitro biosynthetic method in which a suppressortRNA chemically acylated with the desired unnatural amino acid is addedto an in vitro extract capable of supporting protein biosynthesis, hasbeen used to site-specifically incorporate over 100 unnatural aminoacids into a variety of proteins of virtually any size. See, e.g., V. W.Cornish, D. Mendel and P. G. Schultz, Angew. Chem. Int. Ed. Engl., 1995,34:621 (1995); C. J. Noren, S. J. Anthony-Cahill, M. C. Griffith, P. G.Schultz, A general method for site-specific incorporation of unnaturalamino acids into proteins, Science 244:182-188 (1989); and, J. D. Bain,C. G. Glabe, T. A. Dix, A. R. Chamberlin, E. S. Diala, Biosyntheticsite-specific incorporation of a non-natural amino acid into apolypeptide, J. Am. Chem. Soc. 111:8013-8014 (1989). A broad range offunctional groups has been introduced into proteins for studies ofprotein stability, protein folding, enzyme mechanism, and signaltransduction.

An in vivo method, termed selective pressure incorporation, wasdeveloped to exploit the promiscuity of wild-type synthetases. See,e.g., N. Budisa, C. Minks, S. Alefelder, W. Wenger, F. M. Dong, L.Moroder and R. Huber, FASEB J., 13:41 (1999). An auxotrophic strain, inwhich the relevant metabolic pathway supplying the cell with aparticular natural amino acid is switched off, is grown in minimal mediacontaining limited concentrations of the natural amino acid, whiletranscription of the target gene is repressed. At the onset of astationary growth phase, the natural amino acid is depleted and replacedwith the unnatural amino acid analog. Induction of expression of therecombinant protein results in the accumulation of a protein containingthe unnatural analog. For example, using this strategy, o, m andp-fluorophenylalanines have been incorporated into proteins, and exhibittwo characteristic shoulders in the UV spectrum which can be easilyidentified, see, e.g., C. Minks, R. Huber, L. Moroder and N. Budisa,Anal. Biochem., 284:29 (2000); trifluoromethionine has been used toreplace methionine in bacteriophage T4 lysozyme to study its interactionwith chitooligosaccharide ligands by ¹⁹F NMR, see, e.g., H. Duewel, E.Daub, V. Robinson and J. F. Honek, Biochemistry, 36:3404 (1997); andtrifluoroleucine has been incorporated in place of leucine, resulting inincreased thermal and chemical stability of a leucine-zipper protein.See, e.g., Y. Tang, G. Ghirlanda, W. A. Petka, T. Nakajima, W. F.DeGrado and D. A. Tirrell, Angew Chem. Int. Ed. Engl., 40:1494 (2001).Moreover, selenomethionine and telluromethionine are incorporated intovarious recombinant proteins to facilitate the solution of phases inX-ray crystallography. See, e.g., W. A. Hendrickson, J. R. Horton and D.M. Lemaster, EMBO J., 9:1665 (1990); J. O. Boles, K. Lewinski, M.Kunkle, J. D. Odom, B. Dunlap, L. Lebioda and M. Hatada, Nat. Struct.Biol., 1:283 (1994); N. Budisa, B. Steipe, P. Demange, C. Eckerskom, J.Kellermann and R. Huber, Eur. J. Biochem., 230:788 (1995); and, N.Budisa, W. Kambrock, S. Steinbacher, A. Humm, L. Prade, T. Neuefeind, L.Moroder and R. Huber, J. Mol. Biol. 270:616 (1997). Methionine analogswith alkene or alkyne functionalities have also been incorporatedefficiently, allowing for additional modification of proteins bychemical means. See, e.g., J. C. van Hest and D. A. Tirrell, FEBS Lett.,428:68 (1998); J. C. van Hest, K. L. Kiick. and D. A. Tirrell, J. Am.Chem. Soc., 122:1282 (2000); and, K. L. Kuick and D. A. Tirrell,Tetrahedron, 56:9487 (2000); U.S. Pat. No. 6,586,207; U.S. PatentPublication 2002/0042097, which are incorporated by reference herein.

The success of this method depends on the recognition of the unnaturalamino acid analogs by aminoacyl-tRNA synthetases, which, in general,require high selectivity to insure the fidelity of protein translation.One way to expand the scope of this method is to relax the substratespecificity of aminoacyl-tRNA synthetases, which has been achieved in alimited number of cases. For example, replacement of Ala²⁹⁴ by Gly inEscherichia coli phenylalanyl-tRNA synthetase (PheRS) increases the sizeof substrate binding pocket, and results in the acylation of tRNAPhe byp-Cl-phenylalanine (p-Cl-Phe). See, M. Ibba, P. Kast and H. Hennecke,Biochemistry, 33:7107 (1994). An Escherichia coli strain harboring thismutant PheRS allows the incorporation of p-Cl-phenylalanine orp-Br-phenylalanine in place of phenylalanine. See, e.g., M. Ibba and H.Hennecke, FEBS Lett., 364:272 (1995); and, N. Sharma, R. Furter, P. Kastand D. A. Tirrell, FEBS Lett., 467:37 (2000). Similarly, a pointmutation Phe130Ser near the amino acid binding site of Escherichia colityrosyl-tRNA synthetase was shown to allow azatyrosine to beincorporated more efficiently than tyrosine. See, F. Hamano-Takaku, T.Iwama, S. Saito-Yano, K. Takaku, Y. Monden, M. Kitabatake, D. Soll andS, Nishimura, J. Biol. Chem., 275:40324 (2000).

Another strategy to incorporate unnatural amino acids into proteins invivo is to modify synthetases that have proofreading mechanisms. Thesesynthetases cannot discriminate and therefore activate amino acids thatare structurally similar to the cognate natural amino acids. This erroris corrected at a separate site, which deacylates the mischarged aminoacid from the tRNA to maintain the fidelity of protein translation. Ifthe proofreading activity of the synthetase is disabled, structuralanalogs that are misactivated may escape the editing function and beincorporated. This approach has been demonstrated recently with thevalyl-tRNA synthetase (ValRS). See, V. Doring, H. D. Mootz, L. A.Nangle, T. L. Hendrickson, V. de Crecy-Lagard, P. Schimmel and P.Marliere, Science, 292:501 (2001). ValRS can misaminoacylate tRNAValwith Cys, Thr, or aminobutyrate (Abu); these noncognate amino acids aresubsequently hydrolyzed by the editing domain. After random mutagenesisof the Escherichia coli chromosome, a mutant Escherichia coli strain wasselected that has a mutation in the editing site of ValRS. Thisedit-defective ValRS incorrectly charges tRNAVal with Cys. Because Abusterically resembles Cys (—SH group of Cys is replaced with —CH3 inAbu), the mutant ValRS also incorporates Abu into proteins when thismutant Escherichia coli strain is grown in the presence of Abu. Massspectrometric analysis shows that about 24% of valines are replaced byAbu at each valine position in the native protein.

Solid-phase synthesis and semisynthetic methods have also allowed forthe synthesis of a number of proteins containing novel amino acids. Forexample, see the following publications and references cited within,which are as follows: Crick, F. H. C., Barrett, L. Brenner, S.Watts-Tobin, R. General nature of the genetic code for proteins. Nature,192:1227-1232 (1961); Hofmann, K., Bohn, H. Studies on polypeptides.XXAVI. The effect of pyrazole-imidazole replacements on the S-proteinactivating potency of an S-peptide fragment, J. Am. Chem,88(24):5914-5919 (1966); Kaiser, E. T. Synthetic approaches tobiologically active peptides and proteins including enyzmes, Acc ChemRes, 22:47-54 (1989); Nakatsuka, T., Sasaki, T., Kaiser, E. T. Peptidesegment coupling catalyzed by the semisynthetic enzyme thiosubtilisin, JAm Chem Soc, 109:3808-3810 (1987); Schnolzer, M., Kent, S B H.Constructing proteins by dovetailing unprotected synthetic peptides:backbone-engineered HIV protease, Science, 256(5054):221-225 (1992);Chaiken, I. M. Semisynthetic peptides and proteins, CRC Crit. RevBiochem, 11(3):255-301 (1981); Offord, R. E. Protein engineering bychemical means? Protein Eng., 1(3):151-157 (1987); and, Jackson, D. Y.,Burnier, J., Quan, C., Stanley, M., Tom, J., Wells, J. A. A DesignedPeptide Ligase for Total Synthesis of Ribonuclease A with UnnaturalCatalytic Residues, Science, 266(5183):243 (1994).

Chemical modification has been used to introduce a variety of unnaturalside chains, including cofactors, spin labels and oligonucleotides intoproteins in vitro. See, e.g., Corey, D. R., Schultz, P. G. Generation ofa hybrid sequence-specific single-stranded deoxyribonuclease, Science,238(4832):1401-1403 (1987); Kaiser, E. T., Lawrence D. S., Rokita, S. E.The chemical modification of enzymatic specificity, Annu Rev Biochem,54:565-595 (1985); Kaiser, E. T., Lawrence, D. S. Chemical mutation ofenzyme active sites, Science, 226(4674):505-511 (1984); Neet, K. E.,Nanci A, Koshland, D. E. Properties of thiol-subtilisin, J. Biol. Chem.,243(24):6392-6401 (1968); Polgar, L. et M. L. Bender. A new enzymecontaining a synthetically formed active site. Thiol-subtilisin. J. Am.Chem Soc, 88:3153-3154 (1966); and, Pollack, S. J., Nakayama, G.Schultz, P. G. Introduction of nucleophiles and spectroscopic probesinto antibody combining sites, Science, 242(4881):1038-1040 (1988).

Alternatively, biosynthetic methods that employ chemically modifiedaminoacyl-tRNAs have been used to incorporate several biophysical probesinto proteins synthesized in vitro. See the following publications andreferences cited within: Brunner, J. New Photolabeling and crosslinkingmethods, Annu. Rev Biochem, 62:483-514 (1993); and, Krieg, U. C.,Walter, P., Hohnson, A. E. Photocrosslinking of the signal sequence ofnascent preprolactin of the 54-kilodalton polypeptide of the signalrecognition particle, Proc. Natl. Acad. Sci, 83(22):8604-8608 (1986).

Previously, it has been shown that unnatural amino acids can besite-specifically incorporated into proteins in vitro by the addition ofchemically aminoacylated suppressor tRNAs to protein synthesis reactionsprogrammed with a gene containing a desired amber nonsense mutation.Using these approaches, one can substitute a number of the common twentyamino acids with close structural homologues, e.g., fluorophenylalaninefor phenylalanine, using strains auxotropic for a particular amino acid.See, e.g., Noren, C. J., Anthony-Cahill, Griffith, M. C., Schultz, P. G.A general method for site-specific incorporation of unnatural aminoacids into proteins, Science, 244: 182-188 (1989); M. W. Nowak, et al.,Science 268:439-42 (1995); Bain, J. D., Glabe, C. G., Dix, T. A.,Chamberlin, A. R., Diala, E. S. Biosynthetic site-specific Incorporationof a non-natural amino acid into a polypeptide, J. Am. Chem Soc,111:8013-8014 (1989); N. Budisa et al., FASEB J. 13:41-51 (1999);Ellman, J. A., Mendel, D., Anthony-Cahill, S., Noren, C. J., Schultz, P.G. Biosynthetic method for introducing unnatural amino acidssite-specifically into proteins, Methods in Enz., vol. 202, 301-336(1992); and, Mendel, D., Cornish, V. W. & Schultz, P. G. Site-DirectedMutagenesis with an Expanded Genetic Code, Annu Rev Biophys. BiomolStruct. 24, 435-62 (1995).

For example, a suppressor tRNA was prepared that recognized the stopcodon UAG and was chemically aminoacylated with an unnatural amino acid.Conventional site-directed mutagenesis was used to introduce the stopcodon TAG, at the site of interest in the protein gene. See, e.g.,Sayers, J. R., Schmidt, W. Eckstein, F. 5′-3′ Exonucleases inphosphorothioate-based olignoucleotide-directed mutagensis, NucleicAcids Res, 16(3):791-802 (1988). When the acylated suppressor tRNA andthe mutant gene were combined in an in vitro transcription/translationsystem, the unnatural amino acid was incorporated in response to the UAGcodon which gave a protein containing that amino acid at the specifiedposition. Experiments using [³H]-Phe and experiments with α-hydroxyacids demonstrated that only the desired amino acid is incorporated atthe position specified by the UAG codon and that this amino acid is notincorporated at any other site in the protein. See, e.g., Noren, et al,supra; Kobayashi et al., (2003) Nature Structural Biology 10(6):425-432;and, Ellman, J. A., Mendel, D., Schultz, P. G. Site-specificincorporation of novel backbone structures into proteins, Science,255(5041):197-200 (1992).

A tRNA may be aminoacylated with a desired amino acid by any method ortechnique, including but not limited to, chemical or enzymaticaminoacylation.

Aminoacylation may be accomplished by aminoacyl tRNA synthetases or byother enzymatic molecules, including but not limited to, ribozymes. Theterm “ribozyme” is interchangeable with “catalytic RNA.” Cech andcoworkers (Cech, 1987, Science, 236:1532-1539; McCorkle et al., 1987,Concepts Biochem. 64:221-226) demonstrated the presence of naturallyoccurring RNAs that can act as catalysts (ribozymes). However, althoughthese natural RNA catalysts have only been shown to act on ribonucleicacid substrates for cleavage and splicing, the recent development ofartificial evolution of ribozymes has expanded the repertoire ofcatalysis to various chemical reactions. Studies have identified RNAmolecules that can catalyze aminoacyl-RNA bonds on their own(2′)3′-termini (Illangakekare et al., 1995 Science 267:643-647), and anRNA molecule which can transfer an amino acid from one RNA molecule toanother (Lohse et al., 1996, Nature 381:442-444).

U.S. Patent Application Publication 2003/0228593, which is incorporatedby reference herein, describes methods to construct ribozymes and theiruse in aminoacylation of tRNAs with naturally encoded and non-naturallyencoded amino acids. Substrate-immobilized forms of enzymatic moleculesthat can aminoacylate tRNAs, including but not limited to, ribozymes,may enable efficient affinity purification of the aminoacylatedproducts. Examples of suitable substrates include agarose, sepharose,and magnetic beads. The production and use of a substrate-immobilizedform of ribozyme for aminoacylation is described in Chemistry andBiology. 2003, 10:1077-1084 and U.S. Patent Application Publication2003/0228593, which are incorporated by reference herein.

Chemical aminoacylation methods include, but are not limited to, thoseintroduced by Hecht and coworkers (Hecht, S. M. Acc. Chem. Res. 1992,25, 545; Heckler, T. G.; Roesser, J. R.; Xu, C.; Chang, P.; Hecht, S. M.Biochemistry 1988, 27, 7254; Hecht, S. M.; Alford, B. I.; Kuroda, Y.;Kitano, S. J. Biol. Chem. 1978, 253, 4517) and by Schultz, Chamberlin,Dougherty and others (Cornish, V. W.; Mendel, D.; Schultz, P. G. Angew.Chem. Int. Ed. Engl. 1995, 34, 621; Robertson, S. A.; Ellman, J. A.;Schultz, P. G. J. Am. Chem. Soc. 1991, 113, 2722; Noren, C. J.;Anthony-Cahill, S. J.; Griffith, M. C.; Schultz, P. G. Science 1989,244, 182; Bain, J. D.; Glabe, C. G.; Dix, T. A.; Chamberlin, A. R. J.Am. Chem. Soc. 1989, 111, 8013; Bain, J. D. et al. Nature 1992, 356,537; Gallivan, J. P.; Lester, H. A.; Dougherty, D. A. Chem. Biol. 1997,4, 740; Turcatti, et al. J. Biol. Chem. 1996, 271, 19991; Nowak, M. W.et al. Science, 1995, 268, 439; Saks, M. E. et al. J. Biol. Chem. 1996,271, 23169; Hohsaka, T. et al. J. Am. Chem. Soc. 1999, 121, 34), whichare incorporated by reference herein, to avoid the use of synthetases inaminoacylation. Such methods or other chemical aminoacylation methodsmay be used to aminoacylate tRNA molecules.

Methods for generating catalytic RNA may involve generating separatepools of randomized ribozyme sequences, performing directed evolution onthe pools, screening the pools for desirable aminoacylation activity,and selecting sequences of those ribozymes exhibiting desiredaminoacylation activity.

Ribozymes can comprise motifs and/or regions that facilitate acylationactivity, such as a GGU motif and a U-rich region. For example, it hasbeen reported that U-rich regions can facilitate recognition of an aminoacid substrate, and a GGU-motif can form base pairs with the 3′ terminiof a tRNA. In combination, the GGU and motif and U-rich regionfacilitate simultaneous recognition of both the amino acid and tRNAsimultaneously, and thereby facilitate aminoacylation of the 3′ terminusof the tRNA.

Ribozymes can be generated by in vitro selection using a partiallyrandomized r24mini conjugated with tRNA^(Asn) _(CCCG), followed bysystematic engineering of a consensus sequence found in the activeclones. An exemplary ribozyme obtained by this method is termed “Fx3ribozyme” and is described in U.S. Pub. App. No. 2003/0228593, thecontents of which is incorporated by reference herein, acts as aversatile catalyst for the synthesis of various aminoacyl-tRNAs chargedwith cognate non-natural amino acids.

Immobilization on a substrate may be used to enable efficient affinitypurification of the aminoacylated tRNAs. Examples of suitable substratesinclude, but are not limited to, agarose, sepharose, and magnetic beads.Ribozymes can be immobilized on resins by taking advantage of thechemical structure of RNA, such as the 3′-cis-diol on the ribose of RNAcan be oxidized with periodate to yield the corresponding dialdehyde tofacilitate immobilization of the RNA on the resin. Various types ofresins can be used including inexpensive hydrazide resins whereinreductive amination makes the interaction between the resin and theribozyme an irreversible linkage. Synthesis of aminoacyl-tRNAs can besignificantly facilitated by this on-column aminoacylation technique.Kourouklis et al. Methods 2005; 36:239-4 describe a column-basedaminoacylation system.

Isolation of the aminoacylated tRNAs can be accomplished in a variety ofways. One suitable method is to elute the aminoacylated tRNAs from acolumn with a buffer such as a sodium acetate solution with 10 mM EDTA,a buffer containing 50 mMN-(2-hydroxyethyl)piperazine-N′-(3-propanesulfonic acid), 12.5 mM KCl,pH 7.0, 10 mM EDTA, or simply an EDTA buffered water (pH 7.0).

The aminoacylated tRNAs can be added to translation reactions in orderto incorporate the amino acid with which the tRNA was aminoacylated in aposition of choice in a polypeptide made by the translation reaction.Examples of translation systems in which the aminoacylated tRNAs of thepresent invention may be used include, but are not limited to celllysates. Cell lysates provide reaction components necessary for in vitrotranslation of a polypeptide from an input mRNA. Examples of suchreaction components include but are not limited to ribosomal proteins,rRNA, amino acids, tRNAs, GTP, ATP, translation initiation andelongation factors and additional factors associated with translation.Additionally, translation systems may be batch translations orcompartmentalized translation. Batch translation systems combinereaction components in a single compartment while compartmentalizedtranslation systems separate the translation reaction components fromreaction products that can inhibit the translation efficiency. Suchtranslation systems are available commercially.

Further, a coupled transcription/translation system may be used. Coupledtranscription/translation systems allow for both transcription of aninput DNA into a corresponding mRNA, which is in turn translated by thereaction components. An example of a commercially available coupledtranscription/translation is the Rapid Translation System (RTS, RocheInc.). The system includes a mixture containing E. coli lysate forproviding translational components such as ribosomes and translationfactors. Additionally, an RNA polymerase is included for thetranscription of the input DNA into an mRNA template for use intranslation. RTS can use compartmentalization of the reaction componentsby way of a membrane interposed between reaction compartments, includinga supply/waste compartment and a transcription/translation compartment.

Aminoacylation of tRNA may be performed by other agents, including butnot limited to, transferases, polymerases, catalytic antibodies,multi-functional proteins, and the like.

Lu et al. in Mol. Cell. 2001 October; 8(4):759-69 describe a method inwhich a protein is chemically ligated to a synthetic peptide containingunnatural amino acids (expressed protein ligation).

Microinjection techniques have also been use incorporate unnatural aminoacids into proteins. See, e.g., M. W. Nowak, P. C. Kearney, J. R.Sampson, M. E. Saks, C. G. Labarca, S. K. Silverman, W. G. Zhong, J.Thorson, J. N. Abelson, N. Davidson, P. G. Schultz, D. A. Dougherty andH. A. Lester, Science, 268:439 (1995); and, D. A. Dougherty, Curr. Opin.Chem. Biol., 4:645 (2000). A Xenopus oocyte was coinjected with two RNAspecies made in vitro: an mRNA encoding the target protein with a UAGstop codon at the amino acid position of interest and an ambersuppressor tRNA aminoacylated with the desired unnatural amino acid. Thetranslational machinery of the oocyte then inserts the unnatural aminoacid at the position specified by UAG. This method has allowed in vivostructure-function studies of integral membrane proteins, which aregenerally not amenable to in vitro expression systems. Examples includethe incorporation of a fluorescent amino acid into tachykininneurokinin-2 receptor to measure distances by fluorescence resonanceenergy transfer, see, e.g., G. Turcatti, K. Nemeth, M. D. Edgerton, U.Meseth, F. Talabot, M. Peitsch, J. Knowles, H. Vogel and A. Chollet, J.Biol. Chem., 271:19991 (1996); the incorporation of biotinylated aminoacids to identify surface-exposed residues in ion channels, see, e.g.,J. P. Gallivan, H. A. Lester and D. A. Dougherty, Chem. Biol., 4:739(1997); the use of caged tyrosine analogs to monitor conformationalchanges in an ion channel in real time, see, e.g., J. C. Miller, S. K.Silverman, P. M. England, D. A. Dougherty and H. A. Lester, Neuron,20:619 (1998); and, the use of alpha hydroxy amino acids to change ionchannel backbones for probing their gating mechanisms. See, e.g., P. M.England, Y. Zhang, D. A. Dougherty and H. A. Lester, Cell, 96:89 (1999);and, T. Lu, A. Y. Ting, J. Mainland, L. Y. Jan, P. G. Schultz and J.Yang, Nat. Neurosci., 4:239 (2001).

The ability to incorporate unnatural amino acids directly into proteinsin vivo offers a wide variety of advantages including but not limitedto, high yields of mutant proteins, technical ease, the potential tostudy the mutant proteins in cells or possibly in living organisms andthe use of these mutant proteins in therapeutic treatments anddiagnostic uses. The ability to include unnatural amino acids withvarious sizes, acidities, nucleophilicities, hydrophobicities, and otherproperties into proteins can greatly expand our ability to rationallyand systematically manipulate the structures of proteins, both to probeprotein function and create new proteins or organisms with novelproperties.

In one attempt to site-specifically incorporate para-F-Phe, a yeastamber suppressor tRNAPheCUA/phenylalanyl-tRNA synthetase pair was usedin a p-F-Phe resistant, Phe auxotrophic Escherichia coli strain. See,e.g., R. Furter, Protein Sci., 7:419 (1998).

It may also be possible to obtain expression of a GH, e.g., hGHpolynucleotide of the present invention using a cell-free (in-vitro)translational system. Translation systems may be cellular or cell-free,and may be prokaryotic or eukaryotic. Cellular translation systemsinclude, but are not limited to, whole cell preparations such aspermeabilized cells or cell cultures wherein a desired nucleic acidsequence can be transcribed to mRNA and the mRNA translated. Cell-freetranslation systems are commercially available and many different typesand systems are well-known. Examples of cell-free systems include, butare not limited to, prokaryotic lysates such as Escherichia colilysates, and eukaryotic lysates such as wheat germ extracts, insect celllysates, rabbit reticulocyte lysates, rabbit oocyte lysates and humancell lysates. Eukaryotic extracts or lysates may be preferred when theresulting protein is glycosylated, phosphorylated or otherwise modifiedbecause many such modifications are only possible in eukaryotic systems.Some of these extracts and lysates are available commercially (Promega;Madison, Wis.; Stratagene; La Jolla, Calif.; Amersham; ArlingtonHeights, Ill.; GIBCO/BRL; Grand Island, N.Y.). Membranous extracts, suchas the canine pancreatic extracts containing microsomal membranes, arealso available which are useful for translating secretory proteins. Inthese systems, which can include either mRNA as a template (in-vitrotranslation) or DNA as a template (combined in-vitro transcription andtranslation), the in vitro synthesis is directed by the ribosomes.Considerable effort has been applied to the development of cell-freeprotein expression systems. See, e.g., Kim, D. M. and J. R. Swartz,Biotechnology and Bioengineering, 74 :309-316 (2001); Kim, D. M. and J.R. Swartz, Biotechnology Letters, 22, 1537-1542, (2000); Kim, D. M., andJ. R. Swartz, Biotechnology Progress, 16, 385-390, (2000); Kim, D. M.,and J. R. Swartz, Biotechnology and Bioengineering, 66, 180-188, (1999);and Patnaik, R. and J. R. Swartz, Biotechniques 24, 862-868, (1998);U.S. Pat. No. 6,337,191; U.S. Patent Publication No. 2002/0081660; WO00/55353; WO 90/05785, which are incorporated by reference herein.Another approach that may be applied to the expression of GH, e.g., hGHpolypeptides comprising a non-naturally encoded amino acid includes themRNA-peptide fusion technique. See, e.g., R. Roberts and J. Szostak,Proc. Natl. Acad. Sci. (USA) 94:12297-12302 (1997); A. Frankel, et al.,Chemistry & Biology 10:1043-1050 (2003). In this approach, an mRNAtemplate linked to puromycin is translated into peptide on the ribosome.If one or more tRNA molecules has been modified, non-natural amino acidscan be incorporated into the peptide as well. After the last mRNA codonhas been read, puromycin captures the C-terminus of the peptide. If theresulting mRNA-peptide conjugate is found to have interesting propertiesin an in vitro assay, its identity can be easily revealed from the mRNAsequence. In this way, one may screen libraries of GH, e.g., hGHpolypeptides comprising one or more non-naturally encoded amino acids toidentify polypeptides having desired properties. More recently, in vitroribosome translations with purified components have been reported thatpermit the synthesis of peptides substituted with non-naturally encodedamino acids. See, e.g., A. Forster et al., Proc. Natl Acad. Sci. (USA)100:6353 (2003).

Reconstituted translation systems may also be used. Mixtures of purifiedtranslation factors have also been used successfully to translate mRNAinto protein as well as combinations of lysates or lysates supplementedwith purified translation factors such as initiation factor-1 (IF-1),IF-2, IF-3 (α or β), elongation factor T (EF-Tu), or terminationfactors. Cell-free systems may also be coupled transcription/translationsystems wherein DNA is introduced to the system, transcribed into mRNAand the mRNA translated as described in Current Protocols in MolecularBiology (F. M. Ausubel et al. editors, Wiley Interscience, 1993), whichis hereby specifically incorporated by reference. RNA transcribed ineukaryotic transcription system may be in the form of heteronuclear RNA(hnRNA) or 5′-end caps (7-methyl guanosine) and 3′-end poly A tailedmature mRNA, which can be an advantage in certain translation systems.For example, capped mRNAs are translated with high efficiency in thereticulocyte lysate system.

IX. Macromolecular Polymers Coupled to GH, e.g., hGH Polypeptides

Various modifications to the non-natural amino acid polypeptidesdescribed herein can be effected using the compositions, methods,techniques and strategies described herein. These modifications includethe incorporation of further functionality onto the non-natural aminoacid component of the polypeptide, including but not limited to, alabel; a dye; a polymer; a water-soluble polymer; a derivative ofpolyethylene glycol; a photocrosslinker; a radionuclide; a cytotoxiccompound; a drug; an affinity label; a photoaffinity label; a reactivecompound; a resin; a second protein or polypeptide or polypeptideanalog; an antibody or antibody fragment; a metal chelator; a cofactor;a fatty acid; a carbohydrate; a polynucleotide; a DNA; a RNA; anantisense polynucleotide; a saccharide; water-soluble dendrimer; acyclodextrin; an inhibitory ribonucleic acid; a biomaterial; ananoparticle; a spin label; a fluorophore, a metal-containing moiety; aradioactive moiety; a novel functional group; a group that covalently ornoncovalently interacts with other molecules; a photocaged moiety; anactinic radiation excitable moiety; a photoisomerizable moiety; biotin;a derivative of biotin; a biotin analogue; a moiety incorporating aheavy atom; a chemically cleavable group; a photocleavable group; anelongated side chain; a carbon-linked sugar; a redox-active agent; anamino thioacid; a toxic moiety; an isotopically labeled moiety; abiophysical probe; a phosphorescent group; a chemiluminescent group; anelectron dense group; a magnetic group; an intercalating group; achromophore; an energy transfer agent; a biologically active agent; adetectable label; a small molecule; a quantum dot; a nanotransmitter; aradionucleotide; a radiotransmitter; a neutron-capture agent; or anycombination of the above, or any other desirable compound or substance.As an illustrative, non-limiting example of the compositions, methods,techniques and strategies described herein, the following descriptionwill focus on adding macromolecular polymers to the non-natural aminoacid polypeptide with the understanding that the compositions, methods,techniques and strategies described thereto are also applicable (withappropriate modifications, if necessary and for which one of skill inthe art could make with the disclosures herein) to adding otherfunctionalities, including but not limited to those listed above.

A wide variety of macromolecular polymers and other molecules can belinked to GH, e.g., hGH polypeptides of the present invention tomodulate biological properties of the GH, e.g., hGH polypeptide, and/orprovide new biological properties to the GH, e.g., hGH molecule. Thesemacromolecular polymers can be linked to the GH, e.g., hGH polypeptidevia a naturally encoded amino acid, via a non-naturally encoded aminoacid, or any functional substituent of a natural or non-natural aminoacid, or any substituent or functional group added to a natural ornon-natural amino acid. The molecular weight of the polymer may be of awide range, including but not limited to, between about 100 Da and about100,000 Da or more. The molecular weight of the polymer may be betweenabout 100 Da and about 100,000 Da, including but not limited to, 100,000Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da,65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da,8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da,1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200Da, and 100 Da. In some embodiments, the molecular weight of the polymeris between about 100 Da and 50,000 Da. In some embodiments, themolecular weight of the polymer is between about 100 Da and 40,000 Da.In some embodiments, the molecular weight of the polymer is betweenabout 1,000 Da and 40,000 Da. In some embodiments, the molecular weightof the polymer is between about 5,000 Da and 40,000 Da. In someembodiments, the molecular weight of the polymer is between about 10,000Da and 40,000 Da.

The present invention provides substantially homogenous preparations ofpolymer:protein conjugates. “Substantially homogenous” as used hereinmeans that polymer:protein conjugate molecules are observed to begreater than half of the total protein. The polymer:protein conjugatehas biological activity and the present “substantially homogenous”PEGylated GH, e.g., hGH polypeptide preparations provided herein arethose which are homogenous enough to display the advantages of ahomogenous preparation, e.g., ease in clinical application inpredictability of lot to lot pharmacokinetics.

One may also choose to prepare a mixture of polymer:protein conjugatemolecules, and the advantage provided herein is that one may select theproportion of mono-polymer:protein conjugate to include in the mixture.Thus, if desired, one may prepare a mixture of various proteins withvarious numbers of polymer moieties attached (i.e., di-, tri-, tetra-,etc.) and combine said conjugates with the mono-polymer:proteinconjugate prepared using the methods of the present invention, and havea mixture with a predetermined proportion of mono-polymer:proteinconjugates.

The polymer selected may be water soluble so that the protein to whichit is attached does not precipitate in an aqueous environment, such as aphysiological environment. The polymer may be branched or unbranched.For therapeutic use of the end-product preparation, the polymer will bepharmaceutically acceptable.

Examples of polymers include but are not limited to polyalkyl ethers andalkoxy-capped analogs thereof (e.g., polyoxyethylene glycol,polyoxyethylene/propylene glycol, and methoxy or ethoxy-capped analogsthereof, especially polyoxyethylene glycol, the latter is also known aspolyethyleneglycol or PEG); polyvinylpyrrolidones; polyvinylalkylethers; polyoxazolines, polyalkyl oxazolines and polyhydroxyalkyloxazolines; polyacrylamides, polyalkyl acrylamides, and polyhydroxyalkylacrylamides (e.g., polyhydroxypropylmethacrylamide and derivativesthereof); polyhydroxyalkyl acrylates; polysialic acids and analogsthereof; hydrophilic peptide sequences; polysaccharides and theirderivatives, including dextran and dextran derivatives, e.g.,carboxymethyldextran, dextran sulfates, aminodextran; cellulose and itsderivatives, e.g., carboxymethyl cellulose, hydroxyalkyl celluloses;chitin and its derivatives, e.g., chitosan, succinyl chitosan,carboxymethylchitin, carboxymethylchitosan; hyaluronic acid and itsderivatives; starches; alginates; chondroitin sulfate; albumin; pullulanand carboxymethyl pullulan; polyaminoacids and derivatives thereof,e.g., polyglutamic acids, polylysines, polyaspartic acids,polyaspartamides; maleic anhydride copolymers such as: styrene maleicanhydride copolymer, divinylethyl ether maleic anhydride copolymer;polyvinyl alcohols; copolymers thereof; terpolymers thereof; mixturesthereof; and derivatives of the foregoing.

The proportion of polyethylene glycol molecules to protein moleculeswill vary, as will their concentrations in the reaction mixture. Ingeneral, the optimum ratio (in terms of efficiency of reaction in thatthere is minimal excess unreacted protein or polymer) may be determinedby the molecular weight of the polyethylene glycol selected and on thenumber of available reactive groups available. As relates to molecularweight, typically the higher the molecular weight of the polymer, thefewer number of polymer molecules which may be attached to the protein.Similarly, branching of the polymer should be taken into account whenoptimizing these parameters. Generally, the higher the molecular weight(or the more branches) the higher the polymer:protein ratio.

As used herein, and when contemplating PEG:GH, e.g., hGH polypeptideconjugates, the term “therapeutically effective amount” refers to anamount which gives the desired benefit to a patient. The amount willvary from one individual to another and will depend upon a number offactors, including the overall physical condition of the patient and theunderlying cause of the condition to be treated. The amount of GH, e.g.,hGH polypeptide used for therapy gives an acceptable rate of change andmaintains desired response at a beneficial level. A therapeuticallyeffective amount of the present compositions may be readily ascertainedby one of ordinary skill in the art using publicly available materialsand procedures.

The water soluble polymer may be any structural form including but notlimited to linear, forked or branched. Typically, the water solublepolymer is a poly(alkylene glycol), such as poly(ethylene glycol) (PEG),but other water soluble polymers can also be employed. By way ofexample, PEG is used to describe certain embodiments of this invention.

PEG is a well-known, water soluble polymer that is commerciallyavailable or can be prepared by ring-opening polymerization of ethyleneglycol according to methods known to those of ordinary skill in the art(Sandler and Karo, Polymer Synthesis, Academic Press, New York, Vol. 3,pages 138-161). The term “PEG” is used broadly to encompass anypolyethylene glycol molecule, without regard to size or to modificationat an end of the PEG, and can be represented as linked to the GH, e.g.,hGH polypeptide by the formula:XO—(CH₂CH₂O)_(n)—CH₂CH₂—Ywhere n is 2 to 10,000 and X is H or a terminal modification, includingbut not limited to, a C₁₋₄ alkyl, a protecting group, or a terminalfunctional group.

In some cases, a PEG used in the invention terminates on one end withhydroxy or methoxy, i.e., X is H or CH₃ (“methoxy PEG”). Alternatively,the PEG can terminate with a reactive group, thereby forming abifunctional polymer. Typical reactive groups can include those reactivegroups that are commonly used to react with the functional groups foundin the 20 common amino acids (including but not limited to, maleimidegroups, activated carbonates (including but not limited to,p-nitrophenyl ester), activated esters (including but not limited to,N-hydroxysuccinimide, p-nitrophenyl ester) and aldehydes) as well asfunctional groups that are inert to the 20 common amino acids but thatreact specifically with complementary functional groups present innon-naturally encoded amino acids (including but not limited to, azidegroups, alkyne groups). It is noted that the other end of the PEG, whichis shown in the above formula by Y, will attach either directly orindirectly to a GH, e.g., hGH polypeptide via a naturally-occurring ornon-naturally encoded amino acid. For instance, Y may be an amide,carbamate or urea linkage to an amine group (including but not limitedto, the epsilon amine of lysine or the N-terminus) of the polypeptide.Alternatively, Y may be a maleimide linkage to a thiol group (includingbut not limited to, the thiol group of cysteine). Alternatively, Y maybe a linkage to a residue not commonly accessible via the 20 commonamino acids. For example, an azide group on the PEG can be reacted withan alkyne group on the GH, e.g., hGH polypeptide to form a Huisgen[3+2]cycloaddition product. Alternatively, an alkyne group on the PEGcan be reacted with an azide group present in a non-naturally encodedamino acid to form a similar product. In some embodiments, a strongnucleophile (including but not limited to, hydrazine, hydrazide,hydroxylamine, semicarbazide) can be reacted with an aldehyde or ketonegroup present in a non-naturally encoded amino acid to form a hydrazone,oxime or semicarbazone, as applicable, which in some cases can befurther reduced by treatment with an appropriate reducing agent.Alternatively, the strong nucleophile can be incorporated into the GH,e.g., hGH polypeptide via a non-naturally encoded amino acid and used toreact preferentially with a ketone or aldehyde group present in thewater soluble polymer.

Any molecular mass for a PEG can be used as practically desired,including but not limited to, from about 100 Daltons (Da) to 100,000 Daor more as desired (including but not limited to, sometimes 0.1-50 kDaor 10-40 kDa). The molecular weight of PEG may be of a wide range,including but not limited to, between about 100 Da and about 100,000 Daor more. The molecular weight of PEG may be between about 100 Da andabout 100,000 Da, including but not limited to, 100,000 Da, 95,000 Da,90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da,25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000Da, 6,000 Da, 5,000 Da. 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da,800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100 Da. Insome embodiments, the molecular weight of PEG is between about 100 Daand 50,000 Da. In some embodiments, the molecular weight of PEG isbetween about 100 Da and 40,000 Da. In some embodiments, the molecularweight of PEG is between about 1,000 Da and 40,000 Da. In someembodiments, the molecular weight of PEG is between about 5,000 Da and40,000 Da. In some embodiments, the molecular weight of PEG is betweenabout 10,000 Da and 40,000 Da. Branched chain PEGs, including but notlimited to, PEG molecules with each chain having a MW ranging from 1-100kDa (including but not limited to, 1-50 kDa or 5-20 kDa) can also beused. The molecular weight of each chain of the branched chain PEG maybe, including but not limited to, between about 1,000 Da and about100,000 Da or more. The molecular weight of each chain of the branchedchain PEG may be between about 1,000 Da and about 100,000 Da, includingbut not limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da,45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000Da, 3,000 Da, 2,000 Da, and 1,000 Da. In some embodiments, the molecularweight of each chain of the branched chain PEG is between about 1,000 Daand 50,000 Da. In some embodiments, the molecular weight of each chainof the branched chain PEG is between about 1,000 Da and 40,000 Da. Insome embodiments, the molecular weight of each chain of the branchedchain PEG is between about 5,000 Da and 40,000 Da. In some embodiments,the molecular weight of each chain of the branched chain PEG is betweenabout 5,000 Da and 20,000 Da. A wide range of PEG molecules aredescribed in, including but not limited to, the Shearwater Polymers,Inc. catalog, Nektar Therapeutics catalog, incorporated herein byreference.

Generally, at least one terminus of the PEG molecule is available forreaction with the non-naturally-encoded amino acid. For example, PEGderivatives bearing alkyne and azide moieties for reaction with aminoacid side chains can be used to attach PEG to non-naturally encodedamino acids as described herein. If the non-naturally encoded amino acidcomprises an azide, then the PEG will typically contain either an alkynemoiety to effect formation of the [3+2]cycloaddition product or anactivated PEG species (i.e., ester, carbonate) containing a phosphinegroup to effect formation of the amide linkage. Alternatively, if thenon-naturally encoded amino acid comprises an alkyne, then the PEG willtypically contain an azide moiety to effect formation of the [3+2]Huisgen cycloaddition product. If the non-naturally encoded amino acidcomprises a carbonyl group, the PEG will typically comprise a potentnucleophile (including but not limited to, a hydrazide, hydrazine,hydroxylamine, or semicarbazide functionality) in order to effectformation of corresponding hydrazone, oxime, and semicarbazone linkages,respectively. In other alternatives, a reverse of the orientation of thereactive groups described above can be used, i.e., an azide moiety inthe non-naturally encoded amino acid can be reacted with a PEGderivative containing an alkyne.

In some embodiments, the GH, e.g., hGH polypeptide variant with a PEGderivative contains a chemical functionality that is reactive with thechemical functionality present on the side chain of the non-naturallyencoded amino acid.

The invention provides in some embodiments azide- andacetylene-containing polymer derivatives comprising a water solublepolymer backbone having an average molecular weight from about 800 Da toabout 100,000 Da. The polymer backbone of the water-soluble polymer canbe poly(ethylene glycol). However, it should be understood that a widevariety of water soluble polymers including but not limited topoly(ethylene)glycol and other related polymers, including poly(dextran)and poly(propylene glycol), are also suitable for use in the practice ofthis invention and that the use of the term PEG or poly(ethylene glycol)is intended to encompass and include all such molecules. The term PEGincludes, but is not limited to, poly(ethylene glycol) in any of itsforms, including bifunctional PEG, multiarmed PEG, derivatized PEG,forked PEG, branched PEG, pendent PEG (i.e. PEG or related polymershaving one or more functional groups pendent to the polymer backbone),or PEG with degradable linkages therein.

PEG is typically clear, colorless, odorless, soluble in water, stable toheat, inert to many chemical agents, does not hydrolyze or deteriorate,and is generally non-toxic. Poly(ethylene glycol) is considered to bebiocompatible, which is to say that PEG is capable of coexistence withliving tissues or organisms without causing harm. More specifically, PEGis substantially non-immunogenic, which is to say that PEG does not tendto produce an immune response in the body. When attached to a moleculehaving some desirable function in the body, such as a biologicallyactive agent, the PEG tends to mask the agent and can reduce oreliminate any immune response so that an organism can tolerate thepresence of the agent. PEG conjugates tend not to produce a substantialimmune response or cause clotting or other undesirable effects. PEGhaving the formula —CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—, where n is from about3 to about 4000, typically from about 20 to about 2000, is suitable foruse in the present invention. PEG having a molecular weight of fromabout 800 Da to about 100,000 Da are in some embodiments of the presentinvention particularly useful as the polymer backbone. The molecularweight of PEG may be of a wide range, including but not limited to,between about 100 Da and about 100,000 Da or more. The molecular weightof PEG may be between about 100 Da and about 100,000 Da, including butnot limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da,75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da,10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da,3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da,400 Da, 300 Da, 200 Da, and 100 Da. In some embodiments, the molecularweight of PEG is between about 100 Da and 50,000 Da. In someembodiments, the molecular weight of PEG is between about 100 Da and40,000 Da. In some embodiments, the molecular weight of PEG is betweenabout 1,000 Da and 40,000 Da. In some embodiments, the molecular weightof PEG is between about 5,000 Da and 40,000 Da. In some embodiments, themolecular weight of PEG is between about 10,000 Da and 40,000 Da.

The polymer backbone can be linear or branched. Branched polymerbackbones are generally known in the art. Typically, a branched polymerhas a central branch core moiety and a plurality of linear polymerchains linked to the central branch core. PEG is commonly used inbranched forms that can be prepared by addition of ethylene oxide tovarious polyols, such as glycerol, glycerol oligomers, pentaerythritoland sorbitol. The central branch moiety can also be derived from severalamino acids, such as lysine. The branched poly(ethylene glycol) can berepresented in general form as R(—PEG-OH)_(m) in which R is derived froma core moiety, such as glycerol, glycerol oligomers, or pentaerythritol,and m represents the number of arms. Multi-armed PEG molecules, such asthose described in U.S. Pat. Nos. 5,932,462; 5,643,575; 5,229,490;4,289,872; U.S. Pat. Appl. 2003/0143596; WO 96/21469; and WO 93/21259,each of which is incorporated by reference herein in its entirety, canalso be used as the polymer backbone.

Branched PEG can also be in the form of a forked PEG represented byPEG(-YCHZ₂)_(n), where Y is a linking group and Z is an activatedterminal group linked to CH by a chain of atoms of defined length.

Yet another branched form, the pendant PEG, has reactive groups, such ascarboxyl, along the PEG backbone rather than at the end of PEG chains.

In addition to these forms of PEG, the polymer can also be prepared withweak or degradable linkages in the backbone. For example, PEG can beprepared with ester linkages in the polymer backbone that are subject tohydrolysis. As shown below, this hydrolysis results in cleavage of thepolymer into fragments of lower molecular weight:—PEG-CO₂—PEG-+H₂O→PEG-CO₂H+HO—PEG-It is understood by those of ordinary skill in the art that the termpoly(ethylene glycol) or PEG represents or includes all the forms knownin the art including but not limited to those disclosed herein.

Many other polymers are also suitable for use in the present invention.In some embodiments, polymer backbones that are water-soluble, with from2 to about 300 termini, are particularly useful in the invention.Examples of suitable polymers include, but are not limited to, otherpoly(alkylene glycols), such as poly(propylene glycol) (“PPG”),copolymers thereof. (including but not limited to copolymers of ethyleneglycol and propylene glycol), terpolymers thereof, mixtures thereof, andthe like. Although the molecular weight of each chain of the polymerbackbone can vary, it is typically in the range of from about 800 Da toabout 100,000 Da, often from about 6,000 Da to about 80,000 Da. Themolecular weight of each chain of the polymer backbone may be betweenabout 100 Da and about 100,000 Da, including but not limited to, 100,000Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da,65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da,8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da,1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200Da, and 100 Da. In some embodiments, the molecular weight of each chainof the polymer backbone is between about 100 Da and 50,000 Da. In someembodiments, the molecular weight of each chain of the polymer backboneis between about 100 Da and 40,000 Da. In some embodiments, themolecular weight of each chain of the polymer backbone is between about1,000 Da and 40,000 Da. In some embodiments, the molecular weight ofeach chain of the polymer backbone is between about 5,000 Da and 40,000Da. In some embodiments, the molecular weight of each chain of thepolymer backbone is between about 10,000 Da and 40,000 Da.

Those of ordinary skill in the art will recognize that the foregoinglist for substantially water soluble backbones is by no means exhaustiveand is merely illustrative, and that all polymeric materials having thequalities described above are contemplated as being suitable for use inthe present invention.

In some embodiments of the present invention the polymer derivatives are“multi-functional”, meaning that the polymer backbone has at least twotermini, and possibly as many as about 300 termini, functionalized oractivated with a functional group. Multifunctional polymer derivativesinclude, but are not limited to, linear polymers having two termini,each terminus being bonded to a functional group which may be the sameor different.

In one embodiment, the polymer derivative has the structure:X-A-POLY-B—N═N═Nwherein:N═N═N is an azide moiety;B is a linking moiety, which may be present or absent;POLY is a water-soluble non-antigenic polymer;A is a linking moiety, which may be present or absent and which may bethe same as B or different; andX is a second functional group.Examples of a linking moiety for A and B include, but are not limitedto, a multiply-functionalized alkyl group containing up to 18, and maycontain between 1-10 carbon atoms. A heteroatom such as nitrogen, oxygenor sulfur may be included with the alkyl chain. The alkyl chain may alsobe branched at a heteroatom. Other examples of a linking moiety for Aand B include, but are not limited to, a multiply functionalized arylgroup, containing up to 10 and may contain 5-6 carbon atoms. The arylgroup may be substituted with one more carbon atoms, nitrogen, oxygen orsulfur atoms. Other examples of suitable linking groups include thoselinking groups described in U.S. Pat. Nos. 5,932,462; 5,643,575; andU.S. Pat. Appl. Publication 2003/0143596, each of which is incorporatedby reference herein. Those of ordinary skill in the art will recognizethat the foregoing list for linking moieties is by no means exhaustiveand is merely illustrative, and that all linking moieties having thequalities described above are contemplated to be suitable for use in thepresent invention.

Examples of suitable functional groups for use as X include, but are notlimited to, hydroxyl, protected hydroxyl, alkoxyl, active ester, such asN-hydroxysuccinimidyl esters and 1-benzotriazolyl esters, activecarbonate, such as N-hydroxysuccinimidyl carbonates and 1-benzotriazolylcarbonates, acetal, aldehyde, aldehyde hydrates, alkenyl, acrylate,methacrylate, acrylamide, active sulfone, amine, aminooxy, protectedamine, hydrazide, protected hydrazide, protected thiol, carboxylic acid,protected carboxylic acid, isocyanate, isothiocyanate, maleimide,vinylsulfone, dithiopyridine, vinylpyridine, iodoacetamide, epoxide,glyoxals, diones, mesylates, tosylates, tresylate, alkene, ketone, andazide. As is understood by those of ordinary skill in the art, theselected X moiety should be compatible with the azide group so thatreaction with the azide group does not occur. The azide-containingpolymer derivatives may be homobifunctional, meaning that the secondfunctional group (i.e., X) is also an azide moiety, orheterobifunctional, meaning that the second functional group is adifferent functional group.

The term “protected” refers to the presence of a protecting group ormoiety that prevents reaction of the chemically reactive functionalgroup under certain reaction conditions. The protecting group will varydepending on the type of chemically reactive group being protected. Forexample, if the chemically reactive group is an amine or a hydrazide,the protecting group can be selected from the group oftert-butyloxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc). Ifthe chemically reactive group is a thiol, the protecting group can beorthopyridyldisulfide. If the chemically reactive group is a carboxylicacid, such as butanoic or propionic acid, or a hydroxyl group, theprotecting group can be benzyl or an alkyl group such as methyl, ethyl,or tert-butyl. Other protecting groups known in the art may also be usedin the present invention.

Specific examples of terminal functional groups in the literatureinclude, but are not limited to, N-succinimidyl carbonate (see e.g.,U.S. Pat. Nos. 5,281,698, 5,468,478), amine (see, e.g., Buckmann et al.Makromol. Chem. 182:1379 (1981), Zalipsky et al. Eur. Polym. J. 19:1177(1983)), hydrazide (See, e.g., Andresz et al. Makromol. Chem. 179:301(1978)), succinimidyl propionate and succinimidyl butanoate (see, e.g.,Olson et al. in Poly(ethylene glycol) Chemistry & BiologicalApplications, pp 170-181, Harris & Zalipsky Eds., ACS, Washington, D.C.,1997; see also U.S. Pat. No. 5,672,662), succinimidyl succinate (See,e.g., Abuchowski et al. Cancer Biochem. Biophys. 7:175 (1984) andJoppich et al. Makromol. Chem. 180:1381 (1979), succinimidyl ester (see,e.g., U.S. Pat. No. 4,670,417), benzotriazole carbonate (see, e.g., U.S.Pat. No. 5,650,234), glycidyl ether (see, e.g., Pitha et al. Eur. J.Biochem. 94:11 (1979), Elling et al., Biotech. Appl. Biochem. 13:354(1991), oxycarbonylimidazole (see, e.g., Beauchamp, et al., Anal.Biochem. 131:25 (1983), Tondelli et al. J. Controlled Release 1:251(1985)), p-nitrophenyl carbonate (see, e.g., Veronese, et al., Appl.Biochem. Biotech., 11: 141 (1985); and Sartore et al., Appl. Biochem.Biotech., 27:45 (1991)), aldehyde (see, e.g., Harris et al. J. Polym.Sci. Chem. Ed. 22:341 (1984), U.S. Pat. No. 5,824,784, U.S. Pat. No.5,252,714), maleimide (see, e.g., Goodson et al. Biotechnology (NY)8:343 (1990), Romani et al. in Chemistry of Peptides and Proteins 2:29(1984)), and Kogan, Synthetic Comm. 22:2417 (1992)),orthopyridyl-disulfide (see, e.g., Woghiren, et al. Bioconj. Chem. 4:314(1993)), acrylol (see, e.g., Sawhney et al., Macromolecules, 26:581(1993)), vinylsulfone (see, e.g., U.S. Pat. No. 5,900,461). All of theabove references and patents are incorporated herein by reference.

In certain embodiments of the present invention, the polymer derivativesof the invention comprise a polymer backbone having the structure:X—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—N═N═Nwherein:X is a functional group as described above; andn is about 20 to about 4000.In another embodiment, the polymer derivatives of the invention comprisea polymer backbone having the structure:X—CH₂CH₂O—(CH₂CH₂O), —CH₂CH₂—O—(CH₂)_(m)—W—N═N═Nwherein:W is an aliphatic or aromatic linker moiety comprising between 1-10carbon atoms;n is about 20 to about 4000; andX is a functional group as described above. m is between 1 and 10.

The azide-containing PEG derivatives of the invention can be prepared bya variety of methods known in the art and/or disclosed herein. In onemethod, shown below, a water soluble polymer backbone having an averagemolecular weight from about 800 Da to about 100,000 Da, the polymerbackbone having a first terminus bonded to a first functional group anda second terminus bonded to a suitable leaving group, is reacted with anazide anion (which may be paired with any of a number of suitablecounter-ions, including sodium, potassium, tert-butylammonium and soforth). The leaving group undergoes a nucleophilic displacement and isreplaced by the azide moiety, affording the desired azide-containing PEGpolymer.X-PEG-L+N₃ ⁻→X-PEG-N₃

As shown, a suitable polymer backbone for use in the present inventionhas the formula X-PEG-L, wherein PEG is poly(ethylene glycol) and X is afunctional group which does not react with azide groups and L is asuitable leaving group. Examples of suitable functional groups include,but are not limited to, hydroxyl, protected hydroxyl, acetal, alkenyl,amine, aminooxy, protected amine, protected hydrazide, protected thiol,carboxylic acid, protected carboxylic acid, maleimide, dithiopyridine,and vinylpyridine, and ketone. Examples of suitable leaving groupsinclude, but are not limited to, chloride, bromide, iodide, mesylate,tresylate, and tosylate.

In another method for preparation of the azide-containing polymerderivatives of the present invention, a linking agent bearing an azidefunctionality is contacted with a water soluble polymer backbone havingan average molecular weight from about 800 Da to about 100,000 Da,wherein the linking agent bears a chemical functionality that will reactselectively with a chemical functionality on the PEG polymer, to form anazide-containing polymer derivative product wherein the azide isseparated from the polymer backbone by a linking group.

An exemplary reaction scheme is shown below:X—PEG-M+N-linker-N═N═N→PG-X-PEG-linker-N═N═Nwherein:PEG is poly(ethylene glycol) and X is a capping group such as alkoxy ora functional group as described above; andM is a functional group that is not reactive with the azidefunctionality but that will react efficiently and selectively with the Nfunctional group.

Examples of suitable functional groups include, but are not limited to,M being a carboxylic acid, carbonate or active ester if N is an amine; Mbeing a ketone if N is a hydrazide or aminooxy moiety; M being a leavinggroup if N is a nucleophile.

Purification of the crude product may be accomplished by known methodsincluding, but are not limited to, precipitation of the product followedby chromatography, if necessary.

A more specific example is shown below in the case of PEG diamine, inwhich one of the amines is protected by a protecting group moiety suchas tert-butyl-Boc and the resulting mono-protected PEG diamine isreacted with a linking moiety that bears the azide functionality:BocHN—PEG-NH₂+HO₂C—(CH₂)₃—N═N═N

In this instance, the amine group can be coupled to the carboxylic acidgroup using a variety of activating agents such as thionyl chloride orcarbodiimide reagents and N-hydroxysuccinimide or N-hydroxybenzotriazoleto create an amide bond between the monoamine PEG derivative and theazide-bearing linker moiety. After successful formation of the amidebond, the resulting N-tert-butyl-Boc-protected azide-containingderivative can be used directly to modify bioactive molecules or it canbe further elaborated to install other useful functional groups. Forinstance, the N-t-Boc group can be hydrolyzed by treatment with strongacid to generate an omega-amino-PEG-azide. The resulting amine can beused as a synthetic handle to install other useful functionality such asmaleimide groups, activated disulfides, activated esters and so forthfor the creation of valuable heterobifunctional reagents.

Heterobifunctional derivatives are particularly useful when it isdesired to attach different molecules to each terminus of the polymer.For example, the omega-N-amino-N-azido PEG would allow the attachment ofa molecule having an activated electrophilic group, such as an aldehyde,ketone, activated ester, activated carbonate and so forth, to oneterminus of the PEG and a molecule having an acetylene group to theother terminus of the PEG.

In another embodiment of the invention, the polymer derivative has thestructure:X-A—POLY-B—C≡C—Rwherein:R can be either H or an alkyl, alkene, alkyoxy, or aryl or substitutedaryl group;B is a linking moiety, which may be present or absent;POLY is a water-soluble non-antigenic polymer;A is a linking moiety, which may be present or absent and which may bethe same as B or different; andX is a second functional group.

Examples of a linking moiety for A and B include, but are not limitedto, a multiply-functionalized alkyl group containing up to 18, and maycontain between 1-10 carbon atoms. A heteroatom such as nitrogen, oxygenor sulfur may be included with the alkyl chain. The alkyl chain may alsobe branched at a heteroatom. Other examples of a linking moiety for Aand B include, but are not limited to, a multiply functionalized arylgroup, containing up to 10 and may contain 5-6 carbon atoms. The arylgroup may be substituted with one more carbon atoms, nitrogen, oxygen,or sulfur atoms. Other examples of suitable linking groups include thoselinking groups described in U.S. Pat. Nos. 5,932,462 and 5,643,575 andU.S. Pat. Appl. Publication 2003/0143596, each of which is incorporatedby reference herein. Those of ordinary skill in the art will recognizethat the foregoing list for linking moieties is by no means exhaustiveand is intended to be merely illustrative, and that a wide variety oflinking moieties having the qualities described above are contemplatedto be useful in the present invention.

Examples of suitable functional groups for use as X include hydroxyl,protected hydroxyl, alkoxyl, active ester, such as N-hydroxysuccinimidylesters and 1-benzotriazolyl esters, active carbonate, such asN-hydroxysuccinimidyl carbonates and 1-benzotriazolyl carbonates,acetal, aldehyde, aldehyde hydrates, alkenyl, acrylate, methacrylate,acrylamide, active sulfone, amine, aminooxy, protected amine, hydrazide,protected hydrazide, protected thiol, carboxylic acid, protectedcarboxylic acid, isocyanate, isothiocyanate, maleimide, vinylsulfone,dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxals, diones,mesylates, tosylates, and tresylate, alkene, ketone, and acetylene. Aswould be understood, the selected X moiety should be compatible with theacetylene group so that reaction with the acetylene group does notoccur. The acetylene-containing polymer derivatives may behomobifunctional, meaning that the second functional group (i.e., X) isalso an acetylene moiety, or heterobifunctional, meaning that the secondfunctional group is a different functional group.

In another embodiment of the present invention, the polymer derivativescomprise a polymer backbone having the structure:X—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—O—(CH₂)_(m)—C≡CHwherein:X is a functional group as described above;n is about 20 to about 4000; andm is between 1 and 10.Specific examples of each of the heterobifunctional PEG polymers areshown below.

The acetylene-containing PEG derivatives of the invention can beprepared using methods known to those of ordinary skill in the artand/or disclosed herein. In one method, a water soluble polymer backbonehaving an average molecular weight from about 800 Da to about 100,000Da, the polymer backbone having a first terminus bonded to a firstfunctional group and a second terminus bonded to a suitable nucleophilicgroup, is reacted with a compound that bears both an acetylenefunctionality and a leaving group that is suitable for reaction with thenucleophilic group on the PEG. When the PEG polymer bearing thenucleophilic moiety and the molecule bearing the leaving group arecombined, the leaving group undergoes a nucleophilic displacement and isreplaced by the nucleophilic moiety, affording the desiredacetylene-containing polymer.X-PEG-Nu+L-A-C→X-PEG-Nu-A-C≡CR′

As shown, a preferred polymer backbone for use in the reaction has theformula X—PEG-Nu, wherein PEG is poly(ethylene glycol), Nu is anucleophilic moiety and X is a functional group that does not react withNu, L or the acetylene functionality.

Examples of Nu include, but are not limited to, amine, alkoxy, aryloxy,sulfhydryl, imino, carboxylate, hydrazide, aminoxy groups that wouldreact primarily via a SN²-type mechanism. Additional examples of Nugroups include those functional groups that would react primarily via annucleophilic addition reaction. Examples of L groups include chloride,bromide, iodide, mesylate, tresylate, and tosylate and other groupsexpected to undergo nucleophilic displacement as well as ketones,aldehydes, thioesters, olefins, alpha-beta unsaturated carbonyl groups,carbonates and other electrophilic groups expected to undergo additionby nucleophiles.

In another embodiment of the present invention, A is an aliphatic linkerof between 1-10 carbon atoms or a substituted aryl ring of between 6-14carbon atoms. X is a functional group which does not react with azidegroups and L is a suitable leaving group

In another method for preparation of the acetylene-containing polymerderivatives of the invention, a PEG polymer having an average molecularweight from about 800 Da to about 100,000 Da, bearing either a protectedfunctional group or a capping agent at one terminus and a suitableleaving group at the other terminus is contacted by an acetylene anion.

An exemplary reaction scheme is shown below:X-PEG-L+—C≡CR′→X—PEG-C≡CR′wherein:PEG is poly(ethylene glycol) and X is a capping group such as alkoxy ora functional group as described above; andR′ is either H, an alkyl, alkoxy, aryl or aryloxy group or a substitutedalkyl, alkoxyl, aryl or aryloxy group.

In the example above, the leaving group L should be sufficientlyreactive to undergo SN2-type displacement when contacted with asufficient concentration of the acetylene anion. The reaction conditionsrequired to accomplish SN2 displacement of leaving groups by acetyleneanions are known to those of ordinary skill in the art.

Purification of the crude product can usually be accomplished by methodsknown in the art including, but are not limited to, precipitation of theproduct followed by chromatography, if necessary.

Water soluble polymers can be linked to the GH, e.g., hGH polypeptidesof the invention. The water soluble polymers may be linked via anon-naturally encoded amino acid incorporated in the GH, e.g., hGHpolypeptide or any functional group or substituent of a non-naturallyencoded or naturally encoded amino acid, or any functional group orsubstituent added to a non-naturally encoded or naturally encoded aminoacid. Alternatively, the water soluble polymers are linked to a GH,e.g., hGH polypeptide incorporating a non-naturally encoded amino acidvia a naturally-occurring amino acid (including but not limited to,cysteine, lysine or the amine group of the N-terminal residue). In somecases, the GH, e.g., hGH polypeptides of the invention comprise 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or more than 10 non-natural amino acids, whereinone or more non-naturally-encoded amino acid(s) are linked to watersoluble polymer(s) (including but not limited to, PEG and/oroligosaccharides). In some cases, the GH, e.g., hGH polypeptides of theinvention further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than10 naturally-encoded amino acid(s) linked to water soluble polymers. Insome cases, the GH, e.g., hGH polypeptides of the invention comprise oneor more non-naturally encoded amino acid(s) linked to water solublepolymers and one or more naturally-occurring amino acids linked to watersoluble polymers. In some embodiments, the water soluble polymers usedin the present invention enhance the serum half-life of the GH, e.g.,hGH polypeptide relative to the unconjugated form.

The number of water soluble polymers linked to a GH, e.g., hGHpolypeptide (i.e., the extent of PEGylation or glycosylation) of thepresent invention can be adjusted to provide an altered (including butnot limited to, increased or decreased) pharmacologic, pharmacokineticor pharmacodynamic characteristic such as in vivo half-life. In someembodiments, the half-life of GH, e.g., hGH is increased at least about10, 20, 30, 40, 50, 60, 70, 80, 90 percent, 2-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold,15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 25-fold, 30-fold,35-fold, 40-fold, 50-fold, or at least about 100-fold over an unmodifiedpolypeptide.

PEG Derivatives Containing a Strong Nucleophilic Group (i.e., Hydrazide,Hydrazine, Hydroxylamine or Semicarbazide)

In one embodiment of the present invention, a GH, e.g., hGH polypeptidecomprising a carbonyl-containing non-naturally encoded amino acid ismodified with a PEG derivative that contains a terminal hydrazine,hydroxylamine, hydrazide or semicarbazide moiety that is linked directlyto the PEG backbone.

In some embodiments, the hydroxylamine-terminal PEG derivative will havethe structure:RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—O—NH₂where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000 (i.e., average molecular weight is between 5-40 kDa).

In some embodiments, the hydrazine- or hydrazide-containing PEGderivative will have the structure:RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—X—NH—NH₂where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000 and X is optionally a carbonyl group (C═O) that can bepresent or absent.

In some embodiments, the semicarbazide-containing PEG derivative willhave the structure:RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—NH—C(O)—NH—NH₂where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000.

In another embodiment of the invention, a GH, e.g., hGH polypeptidecomprising a carbonyl-containing amino acid is modified with a PEGderivative that contains a terminal hydroxylamine, hydrazide, hydrazine,or semicarbazide moiety that is linked to the PEG backbone by means ofan amide linkage.

In some embodiments, the hydroxylamine-terminal PEG derivatives have thestructure:RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—NH—C(O)(CH₂)_(n)—O—NH₂where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000 (i.e., average molecular weight is between 5-40 kDa).

In some embodiments, the hydrazine- or hydrazide-containing PEGderivatives have the structure:RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—NH—C(O)(CH₂)_(m)—X—NH—NH₂where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, n is100-1,000 and X is optionally a carbonyl group (C═O) that can be presentor absent.

In some embodiments, the semicarbazide-containing PEG derivatives havethe structure:RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—NH—C(O)(CH₂)_(m)—NH—C(O)—NH—NH₂where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000.

In another embodiment of the invention, a GH, e.g., hGH polypeptidecomprising a carbonyl-containing amino acid is modified with a branchedPEG derivative that contains a terminal hydrazine, hydroxylamine,hydrazide or semicarbazide moiety, with each chain of the branched PEGhaving a MW ranging from 10-40 kDa and, may be from 5-20 kDa.

In another embodiment of the invention, a GH, e.g., hGH polypeptidecomprising a non-naturally encoded amino acid is modified with a PEGderivative having a branched structure. For instance, in someembodiments, the hydrazine- or hydrazide-terminal PEG derivative willhave the following structure:[RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—NH—C(O)]₂CH(CH₂)_(m)—X—NH—NH₂where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000, and X is optionally a carbonyl group (C═O) that can bepresent or absent.

In some embodiments, the PEG derivatives containing a semicarbazidegroup will have the structure:[RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—C(O)—NH—CH₂—CH₂]₂CH—X—(CH₂)_(m)—NH—C(O)—NH—NH₂where R is a simple alkyl (methyl, ethyl, propyl, etc.), X is optionallyNH, O, S, C(O) or not present, m is 2-10 and n is 100-1,000.

In some embodiments, the PEG derivatives containing a hydroxylaminegroup will have the structure:[RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—C(O)—NH—CH₂—CH₂]₂CH—X—(CH₂)_(m)—O—NH₂where R is a simple alkyl (methyl, ethyl, propyl, etc.), X is optionallyNH, O, S, C(O) or not present, m is 2-10 and n is 100-1,000.

The degree and sites at which the water soluble polymer(s) are linked tothe hGH polypeptide can modulate the binding of the hGH polypeptide tothe hGH polypeptide receptor at Site 1. In some embodiments, theinvention provides a GH, e.g., hGH, that is linked to at least one PEGby an oxime bond, where the PEG used in the reaction to form the oximebond is a linear, 30 kDa monomethoxy-poly(ethylene glycol)-2-aminooxyethylamine carbamate hydrochloride, as shown in FIG. 19. FIG. 20presents an illustrative example of the synthesis of a linear, 30 kDamonomethoxy-poly(ethylene glycol)-2-aminooxy ethylamine carbamatehydrochloride useful in the synthesis of certain embodiments of theinvention.

By way of example only and not as a limitation on the types or classesof PEG reagents that may be used with the compositions, methods,techniques and strategies described herein, FIG. 21 presents furtherillustrative examples of hydroxylamine-containing PEG reagents that canreact with carbonyl-containing non-natural amino acid polypeptides toform oxime-containing non-natural amino acid polypeptides linked to thePEG group, as well as examples of carbonyl-containing PEG reagents thatcan react with react with oxime-containing non-natural amino acidpolypeptides or hydroxylamine-containing non-natural amino acidpolypeptides to form new oxime-containing non-natural amino acidpolypeptides linked to PEG groups. FIG. 22 presents four illustrativeexamples of synthetic methods for forming hydroxylamine-containing PEGreagents, or protected forms of hydroxylamine-containing PEG reagents,or masked forms of hydroxylamine-containing PEG reagents. FIG. 23presents an illustrative example of synthetic methods for formingamide-linked hydroxylamine-containing PEG reagents, or protected formsof amide-linked hydroxylamine-containing PEG reagents, or masked formsof amide-linked hydroxylamine-containing PEG reagents. FIG. 24 and FIG.25 present an illustrative examples of synthetic methods for formingcarbamate-linked hydroxylamine-containing PEG reagents, or protectedforms of carbamate-linked hydroxylamine-containing PEG reagents, ormasked forms of carbamate-linked hydroxylamine-containing PEG reagents.FIG. 26 presents illustrative examples of synthetic methods for formingsimple hydroxylamine-containing PEG reagents, or protected forms ofsimple hydroxylamine-containing PEG reagents, or masked forms of simplehydroxylamine-containing PEG reagents. Further, FIG. 27 presentsillustrative examples of hydroxylamine-containing reagents that havemultiple branches of linked PEG groups, and further shows the reactionof one such hydroxylamine-containing multi-PEG-branched reagents with acarbonyl-containing non-natural amino acid polypeptide to form anoxime-containing non-natural amino acid polypeptide with a linkedmulti-PEG-branched group.

Further examples of water soluble polymers, e.g., PEGs, useful in theinvention, e.g., PEG modified to be capable of forming an oxime bond,may be found in U.S. Patent Application Nos. 60/638,418; 60/638,527; and60/639,195, entitled “Compositions containing, methods involving, anduses of non-natural amino acids and polypeptides,” filed Dec. 22, 2004,which are incorporated herein by reference in their entirety. Also theyare described in U.S. Patent Application Nos. 60/696,210; 60/696,302;and 60/696,068, entitled “Compositions containing, methods involving,and uses of non-natural amino acids and polypeptides,” filed Jul. 1,2005, which are incorporated herein by reference in their entirety.

The degree and sites at which the water soluble polymer(s) are linked tothe GH, e.g., hGH polypeptide can modulate the binding of the GH, e.g.,hGH polypeptide to the GH, e.g., hGH polypeptide receptor at Site 1. Insome embodiments, the linkages are arranged such that the GH, e.g., hGHpolypeptide binds the GH, e.g., hGH polypeptide receptor at Site 1 witha K_(d) of about 400 nM or lower, with a K_(d) of 150 nM or lower, andin some cases with a K_(d) of 100 nM or lower, as measured by anequilibrium binding assay, such as that described in Spencer et al., J.Biol. Chem., 263:7862-7867 (1988) for GH, e.g., hGH.

Methods and chemistry for activation of polymers as well as forconjugation of peptides are described in the literature and are known inthe art. Commonly used methods for activation of polymers include, butare not limited to, activation of functional groups with cyanogenbromide, periodate, glutaraldehyde, biepoxides, epichlorohydrin,divinylsulfone, carbodiimide, sulfonyl halides, trichlorotriazine, etc.(see, R. F. Taylor, (1991), PROTEIN IMMOBILISATION. FUNDAMENTAL ANDAPPLICATIONS, Marcel Dekker, N.Y.; S. S. Wong, (1992), CHEMISTRY OFPROTEIN CONJUGATION AND CROSSLINKING, CRC Press, Boca Raton; G. T.Hermanson et al., (1993), IMMOBILIZED AFFINITY LIGAND TECHNIQUES,Academic Press, N.Y.; Dunn, R. L., et al., Eds. POLYMERIC DRUGS AND DRUGDELIVERY SYSTEMS, ACS Symposium Series Vol. 469, American ChemicalSociety, Washington, D.C. 1991).

Several reviews and monographs on the functionalization and conjugationof PEG are available. See, for example, Harris, Macromol. Chem. Phys.C25: 325-373 (1985); Scouten, Methods in Enzymology 135: 30-65 (1987);Wong et al., Enzyme Microb. Technol. 14: 866-874 (1992); Delgado et al.,Critical Reviews in Therapeutic Drug Carrier Systems 9: 249-304 (1992);Zalipsky, Bioconjugate Chem. 6: 150-165 (1995).

Methods for activation of polymers can also be found in WO 94/17039,U.S. Pat. No. 5,324,844, WO 94/18247, WO 94/04193, U.S. Pat. No.5,219,564, U.S. Pat. No. 5,122,614, WO 90/13540, U.S. Pat. No.5,281,698, and WO 93/15189, and for conjugation between activatedpolymers and enzymes including but not limited to Coagulation FactorVIII (WO 94/15625), hemoglobin (WO 94/09027), oxygen carrying molecule(U.S. Pat. No. 4,412,989), ribonuclease and superoxide dismutase(Veronese at al., App. Biochem. Biotech. 11: 141-52 (1985)). Allreferences and patents cited are incorporated by reference herein.

PEGylation (i.e., addition of any water soluble polymer) of GH, e.g.,hGH polypeptides containing a non-naturally encoded amino acid, such asp-azido-L-phenylalanine, is carried out by any convenient method. Forexample, GH, e.g., hGH polypeptide is PEGylated with analkyne-terminated MPEG derivative. Briefly, an excess of solidmPEG(5000)-O—CH₂—C≡CH is added, with stirring, to an aqueous solution ofp-azido-L-Phe-containing GH, e.g., hGH polypeptide at room temperature.Typically, the aqueous solution is buffered with a buffer having apK_(a) near the pH at which the reaction is to be carried out (generallyabout pH 4-10). Examples of suitable buffers for PEGylation at pH 7.5,for instance, include, but are not limited to, HEPES, phosphate, borate,TRIS-HCl, EPPS, and TES. The pH is continuously monitored and adjustedif necessary. The reaction is typically allowed to continue for betweenabout 1-48 hours.

The reaction products are subsequently subjected to hydrophobicinteraction chromatography to separate the PEGylated GH, e.g., hGHpolypeptide variants from free mPEG(5000)-O—CH₂—C≡CH and anyhigh-molecular weight complexes of the pegylated GH, e.g., hGHpolypeptide which may form when unblocked PEG is activated at both endsof the molecule, thereby crosslinking GH, e.g., hGH polypeptide variantmolecules. The conditions during hydrophobic interaction chromatographyare such that free mPEG(5000)-O—CH₂—C≡CH flows through the column, whileany crosslinked PEGylated GH, e.g., hGH polypeptide variant complexeselute after the desired forms, which contain one GH, e.g., hGHpolypeptide variant molecule conjugated to one or more PEG groups.Suitable conditions vary depending on the relative sizes of thecross-linked complexes versus the desired conjugates and are readilydetermined by those of ordinary skill in the art. The eluent containingthe desired conjugates is concentrated by ultrafiltration and desaltedby diafiltration.

If necessary, the PEGylated GH, e.g., hGH polypeptide obtained from thehydrophobic chromatography can be purified further by one or moreprocedures known to those of ordinary skill in the art including, butare not limited to, affinity chromatography; anion- or cation-exchangechromatography (using, including but not limited to, DEAE SEPHAROSE);chromatography on silica; reverse phase HPLC; gel filtration (using,including but not limited to, SEPHADEX G-75); hydrophobic interactionchromatography; size-exclusion chromatography, metal-chelatechromatography; ultrafiltration/diafiltration; ethanol precipitation;ammonium sulfate precipitation; chromatofocusing; displacementchromatography; electrophoretic procedures (including but not limited topreparative isoelectric focusing), differential solubility (includingbut not limited to ammonium sulfate precipitation), or extraction.Apparent molecular weight may be estimated by GPC by comparison toglobular protein standards (Preneta, A Z in PROTEIN PURIFICATIONMETHODS, A PRACTICAL APPROACH (Harris & Angal, Eds.) IRL Press 1989,293-306). The purity of the GH, e.g., hGH-PEG conjugate can be assessedby proteolytic degradation (including but not limited to, trypsincleavage) followed by mass spectrometry analysis. Pepinsky RB., et al.,J. Pharmcol. & Exp. Ther. 297(3):1059-66 (2001).

PEGylation (i.e., addition of any water soluble polymer) of GH, e.g.,hGH polypeptides containing a non-naturally encoded amino acidcontaining a carbonyl group, e.g., such as p-acetyl-L-phenylalanine, isalso carried out by any convenient method. As a non-exclusive example, aGH, hGH polypeptide containing a carbonyl-containing non-naturallyencoded amino acid, e.g., p-acetyl-L-phenylalanine, is PEGylated with anaminooxy ethylamine carbamate MPEG derivative of MW about 0.1-100 kDa,or about 1-100 kDa, or about 10-50 kDa, or about 20-40 kDa, or e.g., 30kDa. Briefly, an excess of solid MPEG-oxyamine e.g.,mPEG(30,000)—O—CO—NH—(CH₂)₂—ONH₃ ⁺ (a linear 30 kDamonomethoxy-poly(ethylene glycol)-2-aminooxy ethylamine carbamatehydrochloride, 30K MPEG-oxyamine) is added, with stirring, to an aqueoussolution of p-acetyl-L-phenylalanine-containing GH, e.g., hGHpolypeptide at room temperature. The molar ratio of PEG:GH, e.g., hGHcan be about 2-15, or about 5-10, or about 5, 6, 7, 8, 9 or 10.Typically, the aqueous solution is buffered with a buffer having apK_(a) near the pH at which the reaction is to be carried out (generallyabout pH 2-8). An of a suitable buffer for PEGylation at pH 4.0, forinstance, includes, but is not limited to, a sodium acetate/glycinebuffer adjusted to pH 4.0 by addition of acetic acid. The reaction istypically allowed to continue for between about 1-60 hours, or about10-50 hours, or about 18-48 hours, or about 39-50 hours, at roomtemperature with gentle shaking. PEGylation can be confirmed by SDS gel.

The reaction products are subsequently subjected to purification from,e.g., from free 30K MPEG-oxyamine and any high-molecular weightcomplexes of the PEGylated GH, e.g., hGH polypeptide which may form whenunblocked PEG is activated at both ends of the molecule, therebycrosslinking GH, e.g., hGH polypeptide variant molecules. Any suitablepurification method may be used, e.g., column chromatography such as aSourceQ column run with SourceQ Buffer A and SourceQ Buffer B. Thereaction mixture may be diluted with TRIS base and SourceQ Buffer A andMilliQ water prior to loading on the column. The eluent containing thedesired conjugates can be further concentrated by ultrafiltration anddesalted by diafiltration.

If necessary, the PEGylated GH, e.g., hGH polypeptide obtained from thechromatography can be purified further by one or more procedures knownto those of ordinary skill in the art and described herein (see, e.g.,above). The final PEGylated GH, e.g., hGH polypeptide, may be obtainedat a purity of greater than 50, 60, 70, 80, 90, 95, 99, 99.9, or 99.99%.Purity may be determined by methods known in the art. Exemplarynon-limiting methods of assessing purity include SDS-PAGE, measuring GH,e.g., hGH using Western blot and ELISA assays, Bradford assay, massspectrometry (including, but no limited to, MALDI-TOF),

HPLC methods such as RP HPLC, cation exchange HPLC, and gel filtrationHPLC, and other methods for characterizing proteins known to those ofordinary skill in the art.

A water soluble polymer linked to an amino acid of a GH, e.g., hGHpolypeptide of the invention can be further derivatized or substitutedwithout limitation.

Azide-Containing PEG Derivatives

In another embodiment of the invention, a GH, e.g., hGH polypeptide ismodified with a PEG derivative that contains an azide moiety that willreact with an alkyne moiety present on the side chain of thenon-naturally encoded amino acid. In general, the PEG derivatives willhave an average molecular weight ranging from 1-100 kDa and, in someembodiments, from 10-40 kDa.

In some embodiments, the azide-terminal PEG derivative will have thestructure:RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—N₃where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000 (i.e., average molecular weight is between 5-40 kDa).

In another embodiment, the azide-terminal PEG derivative will have thestructure:RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—NH—C(O)—(CH₂)_(p)—N₃where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, p is2-10 and n is 100-1,000 (i.e., average molecular weight is between 5-40kDa).

In another embodiment of the invention, a GH, e.g., hGH polypeptidecomprising a alkyne-containing amino acid is modified with a branchedPEG derivative that contains a terminal azide moiety, with each chain ofthe branched PEG having a MW ranging from 10-40 kDa and, may be from5-20 kDa. For instance, in some embodiments, the azide-terminal PEGderivative will have the following structure:[RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—NH—C(O)]₂CH(CH₂)_(m)—X—(CH₂)_(p)N₃where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, p is2-10, and n is 100-1,000, and X is optionally an O, N, S or carbonylgroup (C═O), in each case that can be present or absent.Alkyne-Containing PEG Derivatives

In another embodiment of the invention, a GH, e.g., hGH polypeptide ismodified with a PEG derivative that contains an alkyne moiety that willreact with an azide moiety present on the side chain of thenon-naturally encoded amino acid.

In some embodiments, the alkyne-terminal PEG derivative will have thefollowing structure:RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—C≡CHwhere R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000 (i.e., average molecular weight is between 5-40 kDa).

In another embodiment of the invention, a GH, e.g., hGH polypeptidecomprising an alkyne-containing non-naturally encoded amino acid ismodified with a PEG derivative that contains a terminal azide orterminal alkyne moiety that is linked to the PEG backbone by means of anamide linkage.

In some embodiments, the alkyne-terminal PEG derivative will have thefollowing structure:RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—NH—C(O)—(CH₂)_(p)—C≡CHwhere R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, p is2-10 and n is 100-1,000.

In another embodiment of the invention, a GH, e.g., hGH polypeptidecomprising an azide-containing amino acid is modified with a branchedPEG derivative that contains a terminal alkyne moiety, with each chainof the branched PEG having a MW ranging from 10-40 kDa and may be from5-20 kDa. For instance, in some embodiments, the alkyne-terminal PEGderivative will have the following structure:[RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—NH—C(O)]₂CH(CH₂)_(m)—X—(CH₂)_(p)C≡CHwhere R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, p is2-10, and n is 100-1,000, and X is optionally an O, N, S or carbonylgroup (C═O), or not present.Phosphine-Containing PEG Derivatives

In another embodiment of the invention, a GH, e.g., hGH polypeptide ismodified with a PEG derivative that contains an activated functionalgroup (including but not limited to, ester, carbonate) furthercomprising an aryl phosphine group that will react with an azide moietypresent on the side chain of the non-naturally encoded amino acid. Ingeneral, the PEG derivatives will have an average molecular weightranging from 1-100 kDa and, in some embodiments, from 10-40 kDa.

In some embodiments, the PEG derivative will have the structure:

wherein n is 1-10; X can be O, N, S or not present, Ph is phenyl, and Wis a water soluble polymer.

In some embodiments, the PEG derivative will have the structure:

wherein X can be O, N, S or not present, Ph is phenyl, W is a watersoluble polymer and R can be H, alkyl, aryl, substituted alkyl andsubstituted aryl groups. Exemplary R groups include but are not limitedto —CH₂, —C(CH₃)₃, —OR′, —NR′R″, —SR′, -halogen, —C(O)R′, —CONR′R″,—S(O)₂R′, —S(O)₂NR′R″, —CN and —NO₂. R′, R″, R′″ and R″″ eachindependently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, including but notlimited to, aryl substituted with 1-3 halogens, substituted orunsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups.When a compound of the invention includes more than one R group, forexample, each of the R groups is independently selected as are each R′,R″, R′″ and R″″ groups when more than one of these groups is present.When R′ and R″ are attached to the same nitrogen atom, they can becombined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.For example, —NR′R″ is meant to include, but not be limited to,1-pyrrolidinyl and 4-morpholinyl. From the above discussion ofsubstituents, one of skill in the art will understand that the term“alkyl” is meant to include groups including carbon atoms bound togroups other than hydrogen groups, such as haloalkyl (including but notlimited to, —CF₃ and —CH₂CF₃) and acyl (including but not limited to,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).Other PEG Derivatives and General PEGylation Techniques

Other exemplary PEG molecules that may be linked to GH, e.g., hGHpolypeptides, as well as PEGylation methods include those described in,e.g., U.S. Patent Publication No. 2004/0001838; 2002/0052009;2003/0162949; 2004/0013637; 2003/0228274; 2003/0220447; 2003/0158333;2003/0143596; 2003/0114647; 2003/0105275; 2003/0105224; 2003/0023023;2002/0156047; 2002/0099133; 2002/0086939; 2002/0082345; 2002/0072573;2002/0052430; 2002/0040076; 2002/0037949; 2002/0002250; 2001/0056171;2001/0044526; 2001/0021763; U.S. Pat. Nos. 6,646,110; 5,824,778;5,476,653; 5,219,564; 5,629,384; 5,736,625; 4,902,502; 5,281,698;5,122,614; 5,473,034; 5,516,673; 5,382,657; 6,552,167; 6,610,281;6,515,100; 6,461,603; 6,436,386; 6,214,966; 5,990,237; 5,900,461;5,739,208; 5,672,662; 5,446,090; 5,808,096; 5,612,460; 5,324,844;5,252,714; 6,420,339; 6,201,072; 6,451,346; 6,306,821; 5,559,213;5,747,646; 5,834,594; 5,849,860; 5,980,948; 6,004,573; 6,129,912; WO97/32607, EP 229,108, EP 402,378, WO 92/16555, WO 94/04193, WO 94/14758,WO 94/17039, WO 94/18247, WO 94/28024, WO 95/00162, WO 95/11924,WO95/13090, WO 95/33490, WO 96/00080, WO 97/18832, WO 98/41562, WO98/48837, WO 99/32134, WO 99/32139, WO 99/32140, WO 96/40791, WO98/32466, WO 95/06058, EP 439 508, WO 97/03106, WO 96/21469, WO95/13312, EP 921 131, WO 98/05363, EP 809 996, WO 96/41813, WO 96/07670,EP 605 963, EP 510 356, EP 400 472, EP 183 503 and EP 154 316, which areincorporated by reference herein. Any of the PEG molecules describedherein may be used in any form, including but not limited to, singlechain, branched chain, multiarm chain, single functional, bi-functional,multi-functional, or any combination thereof.

Enhancing Affinity for Serum Albumin

Various molecules can also be fused to the GH, e.g., hGH polypeptides ofthe invention to modulate the half-life of GH, e.g., hGH polypeptides inserum. In some embodiments, molecules are linked or fused to GH, e.g.,hGH polypeptides of the invention to enhance affinity for endogenousserum albumin in an animal.

For example, in some cases, a recombinant fusion of a GH, e.g., hGHpolypeptide and an albumin binding sequence is made. Exemplary albuminbinding sequences include, but are not limited to, the albumin bindingdomain from streptococcal protein G (see. e.g., Makrides et al., J.Pharmacol. Exp. Ther. 277:534-542 (1996) and Sjolander et al., J.Immunol. Methods 201:115-123 (1997)), or albumin-binding peptides suchas those described in, e.g., Dennis, et al., J. Biol. Chem.277:35035-35043 (2002).

In other embodiments, the GH, e.g., hGH polypeptides of the presentinvention are acylated with fatty acids. In some cases, the fatty acidspromote binding to serum albumin. See, e.g., Kurtzhals, et al., Biochem.J. 312:725-731 (1995).

In other embodiments, the GH, e.g., hGH polypeptides of the inventionare fused directly with serum albumin (including but not limited to,human serum albumin). Those of skill in the art will recognize that awide variety of other molecules can also be linked to GH, e.g., hGH inthe present invention to modulate binding to serum albumin or otherserum components.

X. Glycosylation of GH, e.g., hGH Polypeptides

The invention includes GH, e.g., hGH polypeptides incorporating one ormore non-naturally encoded amino acids bearing saccharide residues. Thesaccharide residues may be either natural (including but not limited to,N-acetylglucosamine) or non-natural (including but not limited to,3-fluorogalactose). The saccharides may be linked to the non-naturallyencoded amino acids either by an N- or O-linked glycosidic linkage(including but not limited to, N-acetylgalactose-L-serine) or anon-natural linkage (including but not limited to, an oxime or thecorresponding C- or S-linked glycoside).

The saccharide (including but not limited to, glycosyl) moieties can beadded to GH, e.g., hGH polypeptides either in vivo or in vitro. In someembodiments of the invention, a GH, e.g., hGH polypeptide comprising acarbonyl-containing non-naturally encoded amino acid is modified with asaccharide derivatized with an aminooxy group to generate thecorresponding glycosylated polypeptide linked via an oxime linkage. Onceattached to the non-naturally encoded amino acid, the saccharide may befurther elaborated by treatment with glycosyltransferases and otherenzymes to generate an oligosaccharide bound to the GH, e.g., hGHpolypeptide. See, e.g., H. Liu, et al. J. Am. Chem. Soc. 125: 1702-1703(2003).

In some embodiments of the invention, a GH, e.g., hGH polypeptidecomprising a carbonyl-containing non-naturally encoded amino acid ismodified directly with a glycan with defined structure prepared as anaminooxy derivative. One of ordinary skill in the art will recognizethat other functionalities, including azide, alkyne, hydrazide,hydrazine, and semicarbazide, can be used to link the saccharide to thenon-naturally encoded amino acid.

In some embodiments of the invention, a GH, e.g., hGH polypeptidecomprising an azide or alkynyl-containing non-naturally encoded aminoacid can then be modified by, including but not limited to, a Huisgen[3+2]cycloaddition reaction with, including but not limited to, alkynylor azide derivatives, respectively. This method allows for proteins tobe modified with extremely high selectivity.

XI. GH Supergene Family Member Dimers and Multimers

The present invention also provides for GH supergene family membercombinations (including but not limited to GH, e.g., hGH and hGHanalogs) such as homodimers, heterodimers, homomultimers, orheteromultimers (i.e., trimers, tetramers, etc.) where a GH supergenefamily member polypeptide such as GH, e.g., hGH containing one or morenon-naturally encoded amino acids is bound to another GH supergenefamily member or variant thereof or any other polypeptide that is anon-GH supergene family member or variant thereof, either directly tothe polypeptide backbone or via a linker. Due to its increased molecularweight compared to monomers, the GH supergene family member, such as GH,e.g., hGH, dimer or multimer conjugates may exhibit new or desirableproperties, including but not limited to different pharmacological,pharmacokinetic, pharmacodynamic, modulated therapeutic half-life, ormodulated plasma half-life relative to the monomeric GH supergene familymember. In some embodiments, the GH supergene family member, such as GH,e.g., hGH, dimers of the invention will modulate the dimerization of theGH supergene family member receptor. In other embodiments, the GHsupergene family member dimers or multimers of the present inventionwill act as a GH supergene family member receptor antagonist, agonist,or modulator.

In some embodiments, one or more of the GH, e.g., hGH molecules presentin a GH, e.g., hGH containing dimer or multimer comprises anon-naturally encoded amino acid linked to a water soluble polymer thatis present within the Site II binding region. As such, each of the GH,e.g., hGH molecules of the dimer or multimer are accessible for bindingto the GH, e.g., hGH polypeptide receptor via the Site I interface butare unavailable for binding to a second GH, e.g., hGH polypeptidereceptor via the Site II interface. Thus, the GH, e.g., hGH polypeptidedimer or multimer can engage the Site I binding sites of each of twodistinct GH, e.g., hGH polypeptide receptors but, as the GH, e.g., hGHmolecules have a water soluble polymer attached to a non-geneticallyencoded amino acid present in the Site II region, the GH, e.g., hGHpolypeptide receptors cannot engage the Site II region of the GH, e.g.,hGH polypeptide ligand and the dimer or multimer acts as a GH, e.g., hGHpolypeptide antagonist. In some embodiments, one or more of the GH,e.g., hGH molecules present in a GH, e.g., hGH polypeptide containingdimer or multimer comprises a non-naturally encoded amino acid linked toa water soluble polymer that is present within the Site I bindingregion, allowing binding to the Site II region. Alternatively, in someembodiments one or more of the GH, e.g., hGH molecules present in a GH,e.g., hGH polypeptide containing dimer or multimer comprises anon-naturally encoded amino acid linked to a water soluble polymer thatis present at a site that is not within the Site I or Site II bindingregion, such that both are available for binding. In some embodiments acombination of GH, e.g., hGH molecules is used having Site I, Site II,or both available for binding. A combination of GH, e.g., hGH moleculeswherein at least one has Site I available for binding, and at least onehas Site II available for binding may provide molecules having a desiredactivity or property. In addition, a combination of GH, e.g., hGHmolecules having both Site I and Site II available for binding mayproduce a super-agonist GH, e.g., hGH molecule.

In some embodiments, the GH supergene family member polypeptides arelinked directly, including but not limited to, via an Asn-Lys amidelinkage or Cys-Cys disulfide linkage. In some embodiments, the linked GHsupergene family member polypeptides, and/or the linked non-GH supergenefamily member, will comprise different non-naturally encoded amino acidsto facilitate dimerization, including but not limited to, an alkyne inone non-naturally encoded amino acid of a first GH, e.g., hGHpolypeptide and an azide in a second non-naturally encoded amino acid ofa second GH supergene family member polypeptide will be conjugated via aHuisgen [3+2]cycloaddition. Alternatively, a first GH supergene familymember, and/or the linked non-GH supergene family member, polypeptidecomprising a ketone-containing non-naturally encoded amino acid can beconjugated to a second GH supergene family member polypeptide comprisinga hydroxylamine-containing non-naturally encoded amino acid and thepolypeptides are reacted via formation of the corresponding oxime.

Alternatively, the two GH supergene family member polypeptides, and/orthe linked non-GH supergene family member, are linked via a linker. Anyhetero- or homo-bifunctional linker can be used to link the two GHsupergene family members, and/or the linked non-GH supergene familymember, polypeptides, which can have the same or different primarysequence. In some cases, the linker used to tether the GH supergenefamily member, and/or the linked non-GH supergene family member,polypeptides together can be a bifunctional PEG reagent. The linker mayhave a wide range of molecular weight or molecular length. Larger orsmaller molecular weight linkers may be used to provide a desiredspatial relationship or conformation between the GH supergene familymember and the linked entity or between the GH supergene family memberand the receptor for the GH supergene family member, or between thelinked entity and the receptor for the GH supergene family member.Linkers having longer or shorter molecular length may also be used toprovide a desired space or flexibility between the GH supergene familymember and the linked entity, or between the GH supergene family memberand its receptor, or between the linked entity and GH supergene familymember receptor. Similarly, a linker having a particular shape orconformation may be utilized to impart a particular shape orconformation to the GH supergene family member or the linked entity,either before or after the GH supergene family member reaches itstarget. This optimization of the spatial relationship between the GHsupergene family member and the linked entity may provide new,modulated, or desired properties to the molecule.

In some embodiments, the invention provides water-soluble bifunctionallinkers that have a dumbbell structure that includes: a) an azide, analkyne, a hydrazine, a hydrazide, a hydroxylamine, or acarbonyl-containing moiety on at least a first end of a polymerbackbone; and b) at least a second functional group on a second end ofthe polymer backbone. The second functional group can be the same ordifferent as the first functional group. The second functional group, insome embodiments, is not reactive with the first functional group. Theinvention provides, in some embodiments, water-soluble compounds thatcomprise at least one arm of a branched molecular structure. Forexample, the branched molecular structure can be dendritic.

In some embodiments, the invention provides multimers comprising one ormore GH supergene family member, such as GH, e.g., hGH, formed byreactions with water soluble activated polymers that have the structure:R—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—Xwherein n is from about 5 to 3,000, m is 2-10, X can be an azide, analkyne, a hydrazine, a hydrazide, an aminooxy group, a hydroxylamine, anacetyl, or carbonyl-containing moiety, and R is a capping group, afunctional group, or a leaving group that can be the same or differentas X. R can be, for example, a functional group selected from the groupconsisting of hydroxyl, protected hydroxyl, alkoxyl,N-hydroxysuccinimidyl ester, 1-benzotriazolyl ester,N-hydroxysuccinimidyl carbonate, 1-benzotriazolyl carbonate, acetal,aldehyde, aldehyde hydrates, alkenyl, acrylate, methacrylate,acrylamide, active sulfone, amine, aminooxy, protected amine, hydrazide,protected hydrazide, protected thiol, carboxylic acid, protectedcarboxylic acid, isocyanate, isothiocyanate, maleimide, vinylsulfone,dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxals, diones,mesylates, tosylates, and tresylate, alkene, and ketone.XII. Measurement of hGH Polypeptide Activity and Affinity of hGHPolypeptide for the hGH Polypeptide Receptor

The hGH receptor can be prepared as described in McFarland et al.,Science, 245: 494-499 (1989) and Leung, D., et al., Nature, 330:537-543(1987). hGH polypeptide activity can be determined using standard orknown in vitro or in vivo assays. For example, cell lines thatproliferate in the presence of hGH (e.g., a cell line expressing the hGHreceptor or a lactogenic receptor) can be used to monitor hGH receptorbinding. See, e.g., Clark, R., et al., J. Biol. Chem. 271(36):21969(1996); Wada, et al., Mol. Endocrinol. 12:146-156 (1998); Gout, P. W.,et al. Cancer Res. 40, 2433-2436 (1980); WO 99/03887. For anon-PEGylated or PEGYlated hGH polypeptide comprising a non-naturalamino acid, the affinity of the hormone for its receptor can be measuredby using a BIAcore™ biosensor (Pharmacia). See, e.g., U.S. Pat. No.5,849,535; Spencer, S. A., et al., J. Biol. Chem., 263:7862-7867 (1988).In vivo animal models for testing hGH activity include those describedin, e.g., Clark et al., J. Biol. Chem. 271(36):21969-21977 (1996).Assays for dimerization capability of hGH polypeptides comprising one ormore non-naturally encoded amino acids can be conducted as described inCunningham, B., et al., Science, 254:821-825 (1991) and Fuh, G., et al.,Science, 256:1677-1680 (1992). All references and patents cited areincorporated by reference herein.

The compilation of references for assay methodologies is not exhaustive,and those of ordinary skill in the art will recognize other assaysuseful for testing for the desired end result.

XIII. Measurement of Potency, Functional In Vivo Half-Life, andPharmacokinetic Parameters

An important aspect of the invention is the prolonged biologicalhalf-life that is obtained by construction of the hGH polypeptide withor without conjugation of the polypeptide to a water soluble polymermoiety. The rapid decrease of hGH polypeptide serum concentrations hasmade it important to evaluate biological responses to treatment withconjugated and non-conjugated hGH polypeptide and variants thereof. Theconjugated and non-conjugated hGH polypeptide and variants thereof ofthe present invention may have prolonged serum half-lives also aftersubcutaneous or i.v. administration, making it possible to measure by,e.g. ELISA method or by a primary screening assay. ELISA or RIA kitsfrom either BioSource International (Camarillo, Calif.) or DiagnosticSystems Laboratories (Webster, Tex.) may be used. Measurement of in vivobiological half-life is carried out as described herein.

The potency and functional in vivo half-life of an hGH polypeptidecomprising a non-naturally encoded amino acid can be determinedaccording to the protocol described in Clark, R., et al., J. Biol. Chem.271(36):21969-21977 (1996).

Pharmacokinetic parameters for a hGH polypeptide comprising anon-naturally encoded amino acid can be evaluated in normalSprague-Dawley male rats (N=5 animals per treatment group). Animals willreceive either a single dose of 25 ug/rat iv or 50 ug/rat sc, andapproximately 5-7 blood samples will be taken according to a pre-definedtime course, generally covering about 6 hours for a GH, e.g., hGHpolypeptide comprising a non-naturally encoded amino acid not conjugatedto a water soluble polymer and about 4 days for a GH, e.g., hGHpolypeptide comprising a non-naturally encoded amino acid and conjugatedto a water soluble polymer. Pharmacokinetic data for GH, e.g., hGHpolypeptides is well-studied in several species and can be compareddirectly to the data obtained for GH, e.g., hGH polypeptides comprisinga non-naturally encoded amino acid. See Mordenti J., et al., Pharm. Res.8(11):1351-59 (1991) for studies related to GH, e.g., hGH.

Pharmacokinetic parameters can also be evaluated in a primate, e.g.,cynomolgus monkeys. Typically, a single injection is administered eithersubcutaneously or intravenously, and serum GH, e.g., hGH, levels aremonitored over time. See, e.g., Examples, for further description.

In some embodiments, the invention provides a GH, e.g., hGH, having anaverage serum half-life of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 4, 15, 16, or more than 16 hours when administered to amammal subcutaneously. In some embodiments, the invention provides a GH,e.g., hGH, having an average serum half-life of at least about 0.25,0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more than 12 hourswhen administered to a mammal subcutaneously. An “average” serumhalf-life is the mean of at least three animals, or at least fouranimals, or at least five animals, or more than five animals. The mammalis, in some embodiments, is a rat; in some embodiments, the mammal is aprimate, such as a cynomolgus monkey, or such as a human. In someembodiments, the invention provides a PEGylated GH, e.g., a PEGylatedhGH, that has an average serum half-life in a mammal that is at leastabout 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14-,15-16-17-18-, 19-, 20-, 21-, 22-, 23-, 24-, 25-, 30-, 35-, 40-, 45-,50-fold, or more than 50-fold the average serum half-life of the GH,e.g., hGH, in its non-PEGylated form, when administered subcutaneously.In some embodiments, the invention provides a PEGylated GH, e.g., aPEGylated hGH, that has an average serum half-life in a mammal that isat least about 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14-,15-16-17-18-, 19-, 20-, 21-, 22-, 23-, 24-, 25-, 30-, 35-, 40-, 45-,50-fold, or more than 50-fold the average serum half-life of the GH,e.g., hGH, in its non-PEGylated form, when administered intravenously.An “average” serum half-life is the mean of at least three animals, orat least four animals, or at least five animals, or more than fiveanimals. The mammal is, in some embodiments, is a rat; in someembodiments, the mammal is a primate, such as a cynomolgus monkey, orsuch as a human. In some embodiments, the GH is a GH, e.g., hGH. In someembodiments, the growth hormone is linked by a covalent bond to at leastone water-soluble polymer, where the covalent bond is an oxime bond. TheGH can be a GH, e.g., hGH. In some embodiments, the GH, e.g., hGH,includes a non-naturally encoded amino acid, such as acarbonyl-containing non-naturally encoded amino acid. In someembodiments, the non-naturally encoded amino acid is a ketone-containingamino acid, e.g., para-acetylphenylalanine. In some embodiments, the GH,e.g., hGH, contains a non-naturally encoded amino acid, e.g.,para-acetylphenylalanine, substituted at a position in the GH, e.g., hGHcorresponding to amino acid 35 in SEQ ID NO: 2. The water-solublepolymer may be a PEG. Suitable PEGs include linear and branched PEGs;any PEG described herein may be used. In certain embodiments, the PEG isa linear PEG of about 0.1-100 kDa, or about 1-100 kDa, or about 10-50kDa, or about 20-40 kDa, or about 30 kDa. In some embodiments, thepharmaceutical composition contains a GH, e.g., hGH, linked to a 30 kDaPEG by an oxime bond, where the oxime bond is between apara-acetylphenylalanine in the GH located at a position correspondingto amino acid 35 in SEQ ID NO: 2 and the PEG.

The specific activity of GH, e.g., hGH polypeptides in accordance withthis invention can be determined by various assays known in the art. Thebiological activity of the GH, e.g., hGH polypeptide muteins, orfragments thereof, obtained and purified in accordance with thisinvention can be tested by methods described or referenced herein orknown to those of ordinary skill in the art.

XIV. Administration and Pharmaceutical Compositions

The polypeptides or proteins of the invention (including but not limitedto, GH, e.g., hGH, synthetases, proteins comprising one or moreunnatural amino acid, etc.) are optionally employed for therapeuticuses, including but not limited to, in combination with a suitablepharmaceutical carrier. Such compositions, for example, comprise atherapeutically effective amount of the compound, and a pharmaceuticallyacceptable carrier or excipient. Such a carrier or excipient includes,but is not limited to, saline, buffered saline, dextrose, water,glycerol, ethanol, and/or combinations thereof. The formulation is madeto suit the mode of administration. In general, methods of administeringproteins are known to those of ordinary skill in the art and can beapplied to administration of the polypeptides of the invention.

In some embodiments, the invention provides a pharmaceutical compositionthat contains a hormone composition comprising a growth hormone linkedby a covalent bond to at least one water-soluble polymer, where thecovalent bond is an oxime bond; and a pharmaceutically acceptableexcipient. The GH can be a hGH. In some embodiments, the GH, e.g., hGH,includes a non-naturally encoded amino acid, such as acarbonyl-containing non-naturally encoded amino acid. In someembodiments, the non-naturally encoded amino acid is a ketone-containingamino acid, e.g., para-acetylphenylalanine. In some embodiments, the GH,e.g., hGH, contains a non-naturally encoded amino acid, e.g.,para-acetylphenylalanine, substituted at a position in the GH, e.g., hGHcorresponding to amino acid 35 in SEQ ID NO: 2. The water-solublepolymer may be a PEG. Suitable PEGs include linear and branched PEGs;any PEG described herein may be used. In certain embodiments, the PEG isa linear PEG of about 0.1-100 kDa, or about 1-100 kDa, or about 10-50kDa, or about 20-40 kDa, or about 30 kDa. In some embodiments, thepharmaceutical composition contains a GH, e.g., a GH, e.g., hGH, linkedto a 30 kDa PEG by an oxime bond, where the oxime bond is between apara-acetylphenylalanine in the GH located at a position correspondingto amino acid 35 in SEQ ID NO: 2 and the PEG.

Therapeutic compositions comprising one or more polypeptide of theinvention are optionally tested in one or more appropriate in vitroand/or in vivo animal models of disease, to confirm efficacy, tissuemetabolism, and to estimate dosages, according to methods known to thoseof ordinary skill in the art. In particular, dosages can be initiallydetermined by activity, stability or other suitable measures ofunnatural herein to natural amino acid homologues (including but notlimited to, comparison of a GH, e.g., hGH polypeptide modified toinclude one or more unnatural amino acids to a natural amino acid GH,e.g., hGH polypeptide), i.e., in a relevant assay.

Administration is by any of the routes normally used for introducing amolecule into ultimate contact with blood or tissue cells. The unnaturalamino acid polypeptides of the invention are administered in anysuitable manner, optionally with one or more pharmaceutically acceptablecarriers. Suitable methods of administering such polypeptides in thecontext of the present invention to a patient are available, and,although more than one route can be used to administer a particularcomposition, a particular route can often provide a more immediate andmore effective action or reaction than another route.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions of thepresent invention.

hGH polypeptides of the invention, including but not limited toPEGylated hGH, may be administered by any conventional route suitablefor proteins or peptides, including, but not limited to parenterally,e.g. injections including, but not limited to, subcutaneously orintravenously or any other form of injections or infusions. Polypeptidecompositions can be administered by a number of routes including, butnot limited to oral, intravenous, intraperitoneal, intramuscular,transdermal, subcutaneous, topical, sublingual, or rectal means.Compositions comprising non-natural amino acid polypeptides, modified orunmodified, can also be administered via liposomes. Such administrationroutes and appropriate formulations are generally known to those ofskill in the art. The hGH polypeptide comprising a non-naturally encodedamino acid, including but not limited to PEGylated hGH, may be usedalone or in combination with other suitable components such as apharmaceutical carrier.

The GH, e.g., hGH polypeptide comprising a non-natural amino acid, aloneor in combination with other suitable components, can also be made intoaerosol formulations (i.e., they can be “nebulized”) to be administeredvia inhalation. Aerosol formulations can be placed into pressurizedacceptable propellants, such as dichlorodifluoromethane, propane,nitrogen, and the like.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations of hGH can be presented in unit-dose or multi-dosesealed containers, such as ampules and vials.

Parenteral administration and intravenous administration are preferredmethods of administration. In particular, the routes of administrationalready in use for natural amino acid homologue therapeutics (includingbut not limited to, those typically used for EPO, GH, G-CSF, GM-CSF,IFNs, interleukins, antibodies, and/or any other pharmaceuticallydelivered protein), along with formulations in current use, providepreferred routes of administration and formulation for the polypeptidesof the invention.

The dose administered to a patient, in the context of the presentinvention, is sufficient to have a beneficial therapeutic response inthe patient over time, or other appropriate activity, depending on theapplication. The dose is determined by the efficacy of the particularvector, or formulation, and the activity, stability or serum half-lifeof the unnatural amino acid polypeptide employed and the condition ofthe patient, as well as the body weight or surface area of the patientto be treated. The size of the dose is also determined by the existence,nature, and extent of any adverse side-effects that accompany theadministration of a particular vector, formulation, or the like in aparticular patient.

In determining the effective amount of the vector or formulation to beadministered in the treatment or prophylaxis of disease (including butnot limited to, cancers, inherited diseases, diabetes, AIDS, or thelike), the physician evaluates circulating plasma levels, formulationtoxicities, progression of the disease, and/or where relevant, theproduction of anti-unnatural amino acid polypeptide antibodies.

The dose administered, for example, to a 70 kilogram patient, istypically in the range equivalent to dosages of currently-usedtherapeutic proteins, adjusted for the altered activity or serumhalf-life of the relevant composition. The vectors or pharmaceuticalformulations of this invention can supplement treatment conditions byany known conventional therapy, including antibody administration,vaccine administration, administration of cytotoxic agents, naturalamino acid polypeptides, nucleic acids, nucleotide analogues, biologicresponse modifiers, and the like.

For administration, formulations of the present invention areadministered at a rate determined by the LD-50 or ED-50 of the relevantformulation, and/or observation of any side-effects of the unnaturalamino acid polypeptides at various concentrations, including but notlimited to, as applied to the mass and overall health of the patient.Administration can be accomplished via single or divided doses.

If a patient undergoing infusion of a formulation develops fevers,chills, or muscle aches, he/she receives the appropriate dose ofaspirin, ibuprofen, acetaminophen or other pain/fever controlling drug.Patients who experience reactions to the infusion such as fever, muscleaches, and chills are premedicated 30 minutes prior to the futureinfusions with either aspirin, acetaminophen, or, including but notlimited to, diphenhydramine. Meperidine is used for more severe chillsand muscle aches that do not quickly respond to antipyretics andantihistamines. Cell infusion is slowed or discontinued depending uponthe severity of the reaction.

Human GH polypeptides of the invention can be administered directly to amammalian subject. Administration is by any of the routes normally usedfor introducing hGH polypeptide to a subject. The hGH polypeptidecompositions according to embodiments of the present invention includethose suitable for oral, rectal, topical, inhalation (including but notlimited to, via an aerosol), buccal (including but not limited to,sub-lingual), vaginal, parenteral (including but not limited to,subcutaneous, intramuscular, intradermal, intraarticular, intrapleural,intraperitoneal, inracerebral, intraarterial, or intravenous), topical(i.e., both skin and mucosal surfaces, including airway surfaces) andtransdermal administration, although the most suitable route in anygiven case will depend on the nature and severity of the condition beingtreated. Administration can be either local or systemic. Theformulations of compounds can be presented in unit-dose or multi-dosesealed containers, such as ampoules and vials. GH, e.g., hGHpolypeptides of the invention can be prepared in a mixture in a unitdosage injectable form (including but not limited to, solution,suspension, or emulsion) with a pharmaceutically acceptable carrier. GH,e.g., hGH polypeptides of the invention can also be administered bycontinuous infusion (using, including but not limited to, minipumps suchas osmotic pumps), single bolus or slow-release depot formulations.

Formulations suitable for administration include aqueous and non-aqueoussolutions, isotonic sterile solutions, which can contain antioxidants,buffers, bacteriostats, and solutes that render the formulationisotonic, and aqueous and non-aqueous sterile suspensions that caninclude suspending agents, solubilizers, thickening agents, stabilizers,and preservatives. Solutions and suspensions can be prepared fromsterile powders, granules, and tablets of the kind previously described.

Freeze-drying is a commonly employed technique for presenting proteinswhich serves to remove water from the protein preparation of interest.Freeze-drying, or lyophilization, is a process by which the material tobe dried is first frozen and then the ice or frozen solvent is removedby sublimation in a vacuum environment. An excipient may be included inpre-lyophilized formulations to enhance stability during thefreeze-drying process and/or to improve stability of the lyophilizedproduct upon storage. Pikal, M. Biopharm. 3(9)26-30 (1990) and Arakawaet al. Pharm. Res. 8(3):285-291 (1991).

The spray drying of pharmaceuticals is also known to those of ordinaryskill in the art. For example, see Broadhead, J. et al., “The SprayDrying of Pharmaceuticals,” in Drug Dev. Ind. Pharm, 18 (11 & 12),1169-1206 (1992). In addition to small molecule pharmaceuticals, avariety of biological materials have been spray dried and these include:enzymes, sera, plasma, micro-organisms and yeasts. Spray drying is auseful technique because it can convert a liquid pharmaceuticalpreparation into a fine, dustless or agglomerated powder in a one-stepprocess. The basic technique comprises the following four steps: a)atomization of the feed solution into a spray; b) spray-air contact; c)drying of the spray; and d) separation of the dried product from thedrying air. U.S. Pat. Nos. 6,235,710 and 6,001,800, which areincorporated by reference herein, describe the preparation ofrecombinant erythropoietin by spray drying.

The pharmaceutical compositions of the invention may comprise apharmaceutically acceptable carrier, excipient, or stabilizer.Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions(including optional pharmaceutically acceptable carriers, excipients, orstabilizers) of the present invention (see, e.g., Remington'sPharmaceutical Sciences, 17^(th) ed. 1985)).

Suitable carriers include buffers containing succinate, phosphate,borate, HEPES, citrate, histidine or histidine derivatives, imidazole,acetate, bicarbonate, and other organic acids; antioxidants includingbut not limited to, ascorbic acid; low molecular weight polypeptidesincluding but not limited to those less than about 10 residues;proteins, including but not limited to, serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers including but not limited to,polyvinylpyrrolidone; amino acids including but not limited to, glycine,glutamine, asparagine, arginine, histidine or histidine derivatives,methionine, glutamate, or lysine; monosaccharides, disaccharides, andother carbohydrates, including but not limited to, trehalose, sucrose,glucose, mannose, or dextrins; chelating agents including but notlimited to, EDTA; divalent metal ions including but not limited to,zinc, cobalt, or copper; sugar alcohols including but not limited to,mannitol or sorbitol; salt-forming counter ions including but notlimited to, sodium; and/or nonionic surfactants including but notlimited to, Tween™ (including but not limited to, Tween 80 (polysorbate80) and Tween 20 (polysorbate 20), Pluronics™ and other pluronic acids,including but not limited to, and other pluronic acids, including butnot limited to, pluronic acid

F68 (poloxamer 188), or PEG. Suitable surfactants include for examplebut are not limited to polyethers based upon poly(ethyleneoxide)-poly(propylene oxide)-poly(ethylene oxide), i.e., (PEO-PPO-PEO),or poly(propylene oxide)-poly(ethylene oxide)-poly(propylene oxide),i.e., (PPO-PEO-PPO), or a combination thereof. PEO-PPO-PEO andPPO-PEO-PPO are commercially available under the trade names Pluronics™,R-Pluronics™, Tetronics™ and R-Tetronics™ (BASF Wyandotte Corp.,Wyandotte, Mich.) and are further described in U.S. Pat. No. 4,820,352incorporated herein in its entirety by reference. Otherethylene/polypropylene block polymers may be suitable surfactants. Asurfactant or a combination of surfactants may be used to stabilizePEGylated hGH against one or more stresses including but not limited tostress that results from agitation. Some of the above may be referred toas “bulking agents.” Some may also be referred to as “tonicitymodifiers.”

GH, e.g., hGH polypeptides of the invention, including those linked towater soluble polymers such as PEG can also be administered by or aspart of sustained-release systems. Sustained-release compositionsinclude, including but not limited to, semi-permeable polymer matricesin the form of shaped articles, including but not limited to, films, ormicrocapsules. Sustained-release matrices include from biocompatiblematerials such as poly(2-hydroxyethyl methacrylate) (Langer et al., J.Biomed. Mater. Res., 15: 267-277 (1981); Langer, Chem. Tech., 12: 98-105(1982), ethylene vinyl acetate (Langer et al., supra) orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988), polylactides (polylacticacid) (U.S. Pat. No. 3,773,919; EP 58,481), polyglycolide (polymer ofglycolic acid), polylactide co-glycolide (copolymers of lactic acid andglycolic acid) polyanhydrides, copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman et al., Biopolymers, 22, 547-556 (1983),poly(ortho)esters, polypeptides, hyaluronic acid, collagen, chondroitinsulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides,nucleic acids, polyamino acids, amino acids such as phenylalanine,tyrosine, isoleucine, polynucleotides, polyvinyl propylene,polyvinylpyrrolidone and silicone. Sustained-release compositions alsoinclude a liposomally entrapped compound. Liposomes containing thecompound are prepared by methods known per se: DE 3,218,121; Eppstein etal., Proc. Natl. Acad. Sci. U.S.A., 82: 3688-3692 (1985); Hwang et al.,Proc. Natl. Acad. Sci. U.S.A., 77: 4030-4034 (1980); EP 52,322; EP36,676; U.S. Pat. No. 4,619,794; EP 143,949; U.S. Pat. No. 5,021,234;Japanese Pat. Appln. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545;and EP 102,324. All references and patents cited are incorporated byreference herein.

Liposomally entrapped GH, e.g., hGH polypeptides can be prepared bymethods described in, e.g., DE 3,218,121; Eppstein et al., Proc. Natl.Acad. Sci. U.S.A., 82: 3688-3692 (1985); Hwang et al., Proc. Natl. Acad.Sci. U.S.A., 77: 4030-4034 (1980); EP 52,322; EP 36,676; U.S. Pat. No.4,619,794; EP 143,949; U.S. Pat. No. 5,021,234; Japanese Pat. Appln.83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324.Composition and size of liposomes are well known or able to be readilydetermined empirically by one of ordinary skill in the art. Someexamples of liposomes as described in, e.g., Park J W, et al., Proc.Natl. Acad. Sci. USA 92:1327-1331 (1995); Lasic D and Papahadjopoulos D(eds): MEDICAL APPLICATIONS OF LIPOSOMES (1998); Drummond D C, et al.,Liposomal drug delivery systems for cancer therapy, in Teicher B (ed):CANCER DRUG DISCOVERY AND DEVELOPMENT (2002); Park J W, et al., Clin.Cancer Res. 8:1172-1181 (2002); Nielsen U B, et al., Biochim. Biophys.Acta 1591(1-3):109-118 (2002); Mamot C, et al., Cancer Res. 63:3154-3161 (2003). All references and patents cited are incorporated byreference herein.

The dose administered to a patient in the context of the presentinvention should be sufficient to cause a beneficial response in thesubject over time. Generally, the total pharmaceutically effectiveamount of the GH, e.g., hGH polypeptide of the present inventionadministered parenterally per dose is in the range of about 0.01μg/kg/day to about 100 μg/kg, or about 0.05 mg/kg to about 1 mg/kg, ofpatient body weight, although this is subject to therapeutic discretion.The frequency of dosing is also subject to therapeutic discretion, andmay be more frequent or less frequent than the commercially availableGH, e.g., hGH polypeptide products approved for use in humans.Generally, a PEGylated GH, e.g., hGH polypeptide of the invention can beadministered by any of the routes of administration described above. Insome embodiments, the invention provides a composition comprising any ofthe GH, e.g., hGH, described herein in a pharmaceutical composition thatis sufficiently stable for the storage and dosing regimens describedherein. Methods of testing stability are known in the art.

XV. Therapeutic Uses of GH, e.g., h GH Polypeptides of the Invention

The GH, e.g., hGH polypeptides of the invention are useful for treatinga wide range of disorders.

The GH, e.g., hGH agonist polypeptides of the invention may be useful,for example, for treating growth deficiency, immune disorders, and forstimulating heart function. Individuals with growth deficienciesinclude, e.g., individuals with Turner's Syndrome, GH-deficientindividuals (including children), children who experience a slowing orretardation in their normal growth curve about 2-3 years before theirgrowth plate closes (sometimes known as “short normal children”), andindividuals where the insulin-like growth factor-I (IGF-I) response toGH has been blocked chemically (i.e., by glucocorticoid treatment) or bya natural condition such as in adult patients where the IGF-I responseto GH is naturally reduced. The hGH polypeptides of the invention may beuseful for treating individuals with the following conditions: pediatricgrowth hormone deficiency, idiopathic short stature, adult growthhormone deficiency of childhood onset, adult growth hormone deficiencyof adult onset, or secondary growth hormone deficiency. Adults diagnosedwith growth hormone deficiency in adulthood may have had a pituitarytumor or radiation. Conditions including but not limited to, metabolicsyndrome, head injury, obesity, osteoporosis, or depression may resultin growth hormone deficiency-like symptoms in adults.

An agonist GH, e.g., hGH variant may act to stimulate the immune systemof a mammal by increasing its immune function, whether the increase isdue to antibody mediation or cell mediation, and whether the immunesystem is endogenous to the host treated with the GH, e.g., hGHpolypeptide or is transplanted from a donor to the host recipient giventhe GH, e.g., hGH polypeptide (as in bone marrow transplants). “Immunedisorders” include any condition in which the immune system of anindividual has a reduced antibody or cellular response to antigens thannormal, including those individuals with small spleens with reducedimmunity due to drug (e.g., chemotherapeutic) treatments. Examplesindividuals with immune disorders include, e.g., elderly patients,individuals undergoing chemotherapy or radiation therapy, individualsrecovering from a major illness, or about to undergo surgery,individuals with AIDS, Patients with congenital and acquired B-celldeficiencies such as hypogammaglobulinemia, common variedagammaglobulinemia, and selective immunoglobulin deficiencies (e.g., IgAdeficiency, patients infected with a virus such as rabies with anincubation time shorter than the immune response of the patient; andindividuals with hereditary disorders such as diGeorge syndrome.

GH, e.g., hGH antagonist polypeptides of the invention may be useful forthe treatment of gigantism and acromegaly, diabetes and complications(diabetic retinopathy, diabetic neuropathy) arising from diabetes,vascular eye diseases (e.g., involving proliferativeneovascularization), nephropathy, and GH-responsive malignancies.

Vascular eye diseases include, e.g., retinopathy (caused by, e.g.,pre-maturity or sickle cell anemia) and macular degeneration.

GH-responsive malignancies include, e.g., Wilm's tumor, sarcomas (e.g.,osteogenic sarcoma), breast, colon, prostate, and thyroid cancer, andcancers of tissues that express GH receptor mRNA (i.e., placenta,thymus, brain, salivary gland, prostate, bone marrow, skeletal muscle,trachea, spinal cord, retina, lymph node and from Burkitt's lymphoma,colorectal carcinoma, lung carcinoma, lymphoblastic leukemia, andmelanoma).

The GH, e.g., hGH agonist polypeptides of the invention may be useful,for example, for treating chronic renal failure, growth failureassociated with chronic renal insufficiency (CRI), short statureassociated with Turner Syndrome, pediatric Prader-Willi Syndrome (PWS),HIV patients with wasting or cachexia, children born small forgestational age (SGA), obesity, and osteoporosis.

Average quantities of the GH, e.g., hGH may vary and in particularshould be based upon the recommendations and prescription of a qualifiedphysician. The exact amount of GH, e.g., hGH is a matter of preferencesubject to such factors as the exact type of condition being treated,the condition of the patient being treated, as well as the otheringredients in the composition. The invention also provides foradministration of a therapeutically effective amount of another activeagent. The amount to be given may be readily determined by one ofordinary skill in the art based upon therapy with hGH.

Pharmaceutical compositions of the invention may be manufactured in aconventional manner.

In some embodiments the invention provides a method of treatment thatincludes administering to an individual in need of treatment aneffective amount of a hormone composition comprising a growth hormone(GH) linked by covalent bond(s) to at least one water-soluble polymer,wherein the covalent bond(s) is an oxime bond. In some embodiments, themethods include administering to the individual, e.g., human, a GH,e.g., hGH. In some embodiments, the GH, e.g., hGH, includes anon-naturally encoded amino acid, such as a carbonyl-containingnon-naturally encoded amino acid. In some embodiments, the non-naturallyencoded amino acid is a ketone-containing amino acid, e.g.,para-acetylphenylalanine. In some embodiments, the GH, e.g., hGH,contains a non-naturally encoded amino acid, e.g.,para-acetylphenylalanine, substituted at a position in the GH, e.g., hGHcorresponding to amino acid 35 in SEQ ID NO: 2. The water-solublepolymer may be a PEG. Suitable PEGs include linear and branched PEGs;any PEG described herein may be used. In certain embodiments, the PEG isa linear PEG of about 0.1-100 kDa, or about 1-100 kDa, or about 10-50kDa, or about 20-40 kDa, or about 30 kDa. In some embodiments, thepharmaceutical composition contains a GH, e.g., a GH, e.g., hGH, linkedto a 30 kDa PEG by an oxime bond, where the oxime bond is between apara-acetylphenylalanine in the GH located at a position correspondingto amino acid 35 in SEQ ID NO: 2 and the PEG. In some embodiments, theindividual who is treated suffers from pediatric growth hormonedeficiency, idiopathic short stature, adult growth hormone deficiency ofchildhood onset, adult growth hormone deficiency of adult onset, orsecondary growth hormone deficiency.

The GH, e.g., hGH, can be administered to the individual in any suitableform route, dose, frequency, and duration, as described herein and asknown in the art. In some embodiments, the invention provides a methodof treatment that includes administering to an individual in need oftreatment an effective amount of a hormone composition comprising agrowth hormone (GH) linked by covalent bond(s) to at least onewater-soluble polymer, wherein the water-soluble polymer is a linearpolymer, and wherein the hormone composition is given at a frequency ofno more than about once every other day, once every 3, 4, 5, or 6 days,once per week, once per every 8, 9, 10, 11, 12, or 13 days, once per twoweeks, once per every 15, 16, 17, 18, 19, or 20 days, once per threeweeks, once per 22, 23, 24, 25, 26, 27, 28, 29, or 30 days, once permonth, or less than about once per month. It will be appreciated thatfrequency of administration may be altered at the discretion of theindividual or, more typically, the treating professional, and that anycombination of frequencies may be used. In some embodiments, the GHcomposition is administered no more that about once per week, once pertwo weeks, once per three weeks, or once per month. In some embodiments,the GH composition is administered no more that about once per week,once per two weeks, or once per month. In some embodiments, the GHcomposition is administered no more that about once per week. In someembodiments, the GH composition is administered no more that about onceper two weeks. In some embodiments, the GH composition is administeredno more that about once per month.

The invention also provides for administration of a therapeuticallyeffective amount of another active agent along with hGH of the presentinvention. The amount to be given may be readily determined by one ofordinary skill in the art based upon therapy with hGH.

Pharmaceutical compositions of the invention may be manufactured inconventional manner.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1

This example describes one of the many potential sets of criteria forthe selection of preferred sites of incorporation of non-naturallyencoded amino acids into hGH.

This example demonstrates how preferred sites within the hGH polypeptidewere selected for introduction of a non-naturally encoded amino acid.The crystal structure 3HHR, composed of hGH complexed with two moleculesof the extracellular domain of receptor (hGHbp), was used to determinepreferred positions into which one or more non-naturally encoded aminoacids could be introduced. Other hGH structures (e.g. 1AXI) wereutilized to examine potential variation of primary and secondarystructural elements between crystal structure datasets. The coordinatesfor these structures are available from the Protein Data Bank (PDB)(Bernstein et al. J. Mol. Biol. 1997, 112, pp 535) or via The ResearchCollaboratory for Structural Bioinformatics PDB available on the WorldWide Web at rcsb.org. The structural model 3HHR contains the entiremature 22 kDa sequence of hGH with the exception of residues 148-153 andthe C-terminal F191 residue which were omitted due to disorder in thecrystal. Two disulfide bridges are present, formed by C53 and C165 andC182 and C185. Sequence numbering used in this example is according tothe amino acid sequence of mature hGH (22 kDa variant) shown in SEQ IDNO:2.

The following criteria were used to evaluate each position of hGH forthe introduction of a non-naturally encoded amino acid: the residue (a)should not interfere with binding of either hGHbp based on structuralanalysis of 3HHR, 1AXI, and 1HWG (crystallographic structures of hGHconjugated with hGHbp monomer or dimer), b) should not be affected byalanine or homolog scanning mutagenesis (Cunningham et al. Science(1989) 244:1081-1085 and Cunningham et al. Science (1989)243:1330-1336), (c) should be surface exposed and exhibit minimal vander Waals or hydrogen bonding interactions with surrounding residues,(d) should be either deleted or variable in hGH variants (e.g. Tyr35,Lys38, Phe92, Lys140), (e) would result in conservative changes uponsubstitution with a non-naturally encoded amino acid and (f) could befound in either highly flexible regions (including but not limited to CDloop) or structurally rigid regions (including but not limited to HelixB). In addition, further calculations were performed on the hGHmolecule, utilizing the Cx program (Pintar et al. (2002) Bioinformatics,18, pp 980) to evaluate the extent of protrusion for each protein atom.As a result, in some embodiments, one or more non-naturally encodedencoded amino acids are incorporated at, but not limited to, one or moreof the following positions of hGH: before position 1 (i.e. at theN-terminus), 1, 2, 3, 4, 5, 8, 9, 11, 12, 15, 16, 19, 22, 29, 30, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 52,55, 57, 59, 65, 66, 69, 70, 71, 74, 88, 91, 92, 94, 95, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 111, 112, 113, 115, 116,119, 120, 122, 123, 126, 127, 129, 130, 131, 132, 133, 134, 135, 136,137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 158, 159, 161, 168, 172, 183, 184, 185,186, 187, 188, 189, 190, 191, 192 (i.e., at the carboxyl terminus of theprotein) (SEQ ID NO: 2 or the corresponding amino acids in SEQ ID NO: 1or 3).

In some embodiments, one or more non-naturally encoded amino acids aresubstituted at one or more of the following positions: 29, 30, 33, 34,35, 37, 39, 40, 49, 57, 59, 66, 69, 70, 71, 74, 88, 91, 92, 94, 95, 98,99, 101, 103, 107, 108, 111, 122, 126, 129, 130, 131, 133, 134, 135,136, 137, 139, 140, 141, 142, 143, 145, 147, 154, 155, 156, 159, 183,186, and 187 (SEQ ID NO: 2 or the corresponding amino acids of SEQ IDNO: 1 or 3).

In some embodiments, one or more non-naturally encoded amino acids aresubstituted at one or more of the following positions: 29, 33, 35, 37,39, 49, 57, 69, 70, 71, 74, 88, 91, 92, 94, 95, 98, 99, 101, 103, 107,108, 111, 129, 130, 131, 133, 134, 135, 136, 137, 139, 140, 141, 142,143, 145, 147, 154, 155, 156, 186, and 187 (SEQ ID NO: 2 or thecorresponding amino acids of SEQ ID NO: 1 or 3).

In some embodiments, one or more non-naturally encoded amino acids aresubstituted at one or more of the following positions: 35, 88, 91, 92,94, 95, 99, 101, 103, 111, 131, 133, 134, 135, 136, 139, 140, 143, 145,and 155 (SEQ ID NO: 2 or the corresponding amino acids of SEQ ID NO: 1or 3).

In some embodiments, one or more non-naturally encoded amino acids aresubstituted at one or more of the following positions: 30, 74, 103 (SEQID NO: 2 or the corresponding amino acids of SEQ ID NO: 1 or 3). In someembodiments, one or more non-naturally encoded amino acids aresubstituted at one or more of the following positions: 35, 92, 143, 145(SEQ ID NO: 2 or the corresponding amino acids of SEQ ID NO: 1 or 3).

In some embodiments, the non-naturally occurring amino acid at one ormore of these positions is linked to a water soluble polymer, includingbut not limited to, positions: before position 1 (i.e. at theN-terminus), 1, 2, 3, 4, 5, 8, 9, 11, 12, 15, 16, 19, 22, 29, 30, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 52,55, 57, 59, 65, 66, 69, 70, 71, 74, 88, 91, 92, 94, 95, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 111, 112, 113, 115, 116,119, 120, 122, 123, 126, 127, 129, 130, 131, 132, 133, 134, 135, 136,137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 158, 159, 161, 168, 172, 183, 184, 185,186, 187, 188, 189, 190, 191, 192 (i.e., at the carboxyl terminus of theprotein) (SEQ ID NO: 2 or the corresponding amino acids of SEQ ID NO: 1or 3). In some embodiments, the non-naturally occurring amino acid atone or more of these positions is linked to a water soluble polymer,including but not limited to, positions: 29, 30, 33, 34, 35, 37, 39, 40,49, 57, 59, 66, 69, 70, 71, 74, 88, 91, 92, 94, 95, 98, 99, 101, 103,107, 108, 111, 122, 126, 129, 130, 131, 133, 134, 135, 136, 137, 139,140, 141, 142, 143, 145, 147, 154, 155, 156, 159, 183, 186, and 187 (SEQID NO: 2 or the corresponding amino acids of SEQ ID NO: 1 or 3).

In some embodiments, the non-naturally occurring amino acid at one ormore of these positions is linked to a water soluble polymer, includingbut not limited to, positions: 29, 33, 35, 37, 39, 49, 57, 69, 70, 71,74, 88, 91, 92, 94, 95, 98, 99, 101, 103, 107, 108, 111, 129, 130, 131,133, 134, 135, 136, 137, 139, 140, 141, 142, 143, 145, 147, 154, 155,156, 186, and 187 (SEQ ID NO: 2 or the corresponding amino acids of SEQID NO: 1 or 3).

In some embodiments, the non-naturally occurring amino acid at one ormore of these positions is linked to a water soluble polymer, includingbut not limited to, positions: 35, 88, 91, 92, 94, 95, 99, 101, 103,111, 131, 133, 134, 135, 136, 139, 140, 143, 145, and 155 (SEQ ID NO: 2or the corresponding amino acids of SEQ ID NO: 1 or 3).

In some embodiments, the non-naturally occurring amino acid at one ormore of these positions is linked to a water soluble polymer, includingbut not limited to, positions: 30, 74, 103 (SEQ ID NO: 2 or thecorresponding amino acids of SEQ ID NO: 1 or 3). In some embodiments,the non-naturally occurring amino acid at one or more of these positionsis linked to a water soluble polymer: 30, 35, 74, 92, 103, 143, 145 (SEQID NO: 2 or the corresponding amino acids of SEQ ID NO: 1 or 3). In someembodiments, the non-naturally occurring amino acid at one or more ofthese positions is linked to a water soluble polymer: 35, 92, 143, 145(SEQ ID NO: 2 or the corresponding amino acids of SEQ ID NO: 1 or 3).

Some sites for generation of an hGH antagonist include: 1, 2, 3, 4, 5,8, 9, 11, 12, 15, 16, 19, 22, 103, 109, 112, 113, 115, 116, 119, 120,123, 127, or an addition before position 1, or any combination thereof(SEQ ID NO: 2, or the corresponding amino acid in SEQ ID NO: 1, 3, orany other GH sequence). These sites were chosen utilizing criteria(c)-(e) of the agonist design. The antagonist design may also includesite-directed modifications of site I residues to increase bindingaffinity to hGHbp.

Example 2

This example details cloning and expression of a hGH polypeptideincluding a non-naturally encoded amino acid in E. coli. This examplealso describes one method to assess the biological activity of modifiedhGH polypeptides.

Methods for cloning hGH and fragments thereof are detailed in U.S. Pat.Nos. 4,601,980; 4,604,359; 4,634,677; 4,658,021; 4,898,830; 5,424,199;and 5,795,745, which are incorporated by reference herein. cDNA encodingthe full length hGH or the mature form of hGH lacking the N-terminalsignal sequence are shown in SEQ ID NO: 21 and SEQ ID NO: 22respectively.

An introduced translation system that comprises an orthogonal tRNA(O-tRNA) and an orthogonal aminoacyl tRNA synthetase (O—RS) is used toexpress hGH containing a non-naturally encoded amino acid. The O—RSpreferentially aminoacylates the O-tRNA with a non-naturally encodedamino acid. In turn the translation system inserts the non-naturallyencoded amino acid into hGH, in response to an encoded selector codon.TABLE 2 O-RS and O-tRNA sequences. SEQ ID NO: 4 M. jannaschiimtRNA_(CUA) ^(Tyr) tRNA SEQ ID NO: 5 HLAD03; an optimized ambersupressor tRNA tRNA SEQ ID NO: 6 HL325A; an optimized AGGA frameshiftsupressor tRNA tRNA SEQ ID NO: 7 Aminoacyl tRNA synthetase for theincorporation of p-azido-L-phenylalanine RS p-Az-PheRS(6) SEQ ID NO: 8Aminoacyl tRNA synthetase for the incorporation ofp-benzoyl-L-phenylalanine RS p-BpaRS(1) SEQ ID NO: 9 Aminoacyl tRNAsynthetase for the incorporation of propargyl-phenylalanine RSPropargyl-PheRS SEQ ID NO: 10 Aminoacyl tRNA synthetase for theincorporation of propargyl-phenylalanine RS Propargyl-PheRS SEQ ID NO:11 Aminoacyl tRNA synthetase for the incorporation ofpropargyl-phenylalanine RS Propargyl-PheRS SEQ ID NO: 12 Aminoacyl tRNAsynthetase for the incorporation of p-azido-phenylalanine RSp-Az-PheRS(1) SEQ ID NO: 13 Aminoacyl tRNA synthetase for theincorporation of p-azido-phenylalanine RS p-Az-PheRS(3) SEQ ID NO: 14Aminoacyl tRNA synthetase for the incorporation of p-azido-phenylalanineRS p-Az-PheRS(4) SEQ ID NO: 15 Aminoacyl tRNA synthetase for theincorporation of p-azido-phenylalanine RS p-Az-PheRS(2) SEQ ID NO: 16Aminoacyl tRNA synthetase for the incorporation ofp-acetyl-phenylalanine (LW1) RS SEQ ID NO: 17 Aminoacyl tRNA synthetasefor the incorporation of p-acetyl-phenylalanine (LW5) RS SEQ ID NO: 18Aminoacyl tRNA synthetase for the incorporation ofp-acetyl-phenylalanine (LW6) RS SEQ ID NO: 19 Aminoacyl tRNA synthetasefor the incorporation of p-azido-phenylalanine (AzPheRS-5) RS SEQ ID NO:20 Aminoacyl tRNA synthetase for the incorporation ofp-azido-phenylalanine (AzPheRS-6) RS

The transformation of E. coli with plasmids containing the modified hGHgene and the orthogonal aminoacyl tRNA synthetase/tRNA pair (specificfor the desired non-naturally encoded amino acid) allows thesite-specific incorporation of non-naturally encoded amino acid into thehGH polypeptide. The transformed E. coli, grown at 37° C. in mediacontaining between 0.01-100 mM of the particular non-naturally encodedamino acid, expresses modified hGH with high fidelity and efficiency.The His-tagged hGH containing a non-naturally encoded amino acid isproduced by the E. coli host cells as inclusion bodies or aggregates.The aggregates are solubilized and affinity purified under denaturingconditions in 6M guanidine HCl. Refolding is performed by dialysis at 4°C. overnight in 50 mM TRIS-HCl, pH8.0, 40 μM CuSO₄, and 2% (w/v)Sarkosyl. The material is then dialyzed against 20 mM TRIS-HCl, pH 8.0,100 mM NaCl, 2mM CaCl₂, followed by removal of the His-tag. See Boisselet al., (1993) J. Bio. Chem. 268:15983-93. Methods for purification ofhGH are known to those of ordinary skill in the art and are confirmed bySDS-PAGE, Western Blot analyses, or electrospray-ionization ion trapmass spectrometry and the like.

FIG. 6 is an SDS-PAGE of purified hGH polypeptides. The His-taggedmutant hGH proteins were purified using the ProBond Nickel-ChelatingResin (Invitrogen, Carlsbad, Calif.) via the standard His-tagged proteinpurification procedures provided by the manufacturer, followed by ananion exchange column prior to loading on the gel. Lane 1 shows themolecular weight marker, and lane 2 represents N-His hGH withoutincorporation of a non-natural amino acid. Lanes 3-10 contain N-His hGHmutants comprising the non-natural amino acid p-acetyl-phenylalanine ateach of the positions Y35, F92, Y111, G131, R134, K140, Y143, and K145,respectively.

To further assess the biological activity of modified hGH polypeptides,an assay measuring a downstream marker of hGH's interaction with itsreceptor was used. The interaction of hGH with its endogenously producedreceptor leads to the tyrosine phosphorylation of a signal transducerand activator of transcription family member, STAT5, in the human IM-9lymphocyte cell line. Two forms of STAT5, STAT5A and STAT5B wereidentified from an IM-9 cDNA library. See, e.g., Silva et al., Mol.Endocrinol. (1996) 10(5):508-518. The human growth hormone receptor onIM-9 cells is selective for human growth hormone as neither rat growthhormone nor human prolactin resulted in detectable STAT5phosphorylation. Importantly, rat GHR (L43R) extra cellular domain andthe G120R bearing hGH compete effectively against hGH stimulated pSTAT5phoshorylation.

IM-9 cells were stimulated with hGH polypeptides of the presentinvention. The human IM-9 lymphocytes were purchased from ATCC(Manassas, Va.) and grown in RPMI 1640 supplemented with sodiumpyruvate, penicillin, streptomycin (Invitrogen, Carlsbad, San Diego) and10% heat inactivated fetal calf serum (Hyclone, Logan, Utah). The IM-9cells were starved overnight in assay media (phenol-red free RPMI, 10 mMHepes, 1% heat inactivated charcoal/dextran treated FBS, sodiumpyruvate, penicillin and streptomycin) before stimulation with a12-point dose range of hGH polypeptides for 10 min at 37° C. Stimulatedcells were fixed with 1% formaldehyde before permeabilization with 90%ice-cold methanol for 1 hour on ice. The level of STAT5 phosphorylationwas detected by intra-cellular staining with a primary phospho-STAT5antibody (Cell Signaling Technology, Beverly, Mass.) at room temperaturefor 30 min followed by a PE-conjugated secondary antibody. Sampleacquisition was performed on the FACS Array with acquired data analyzedon the Flowjo software (Tree Star Inc., Ashland, Oreg.). EC₅₀ valueswere derived from dose response curves plotted with mean fluorescentintensity (MFI) against protein concentration utilizing SigmaPlot.

Table 3 below summarizes the IM-9 data generated with mutant hGHpolypeptides. Various hGH polypeptides with a non-natural amino acidsubstitution at different positions were tested with human IM-9 cells asdescribed. Specifically, FIG. 7, Panel A shows the IM-9 data for aHis-tagged hGH polypeptide, and FIG. 7, Panel B shows the IM-9 data forHis-tagged hGH comprising the non-natural amino acidp-acetyl-phenylalanine substitution for Y143. The same assay was used toassess biological activity of hGH polypeptides comprising a non-naturalamino acid that is PEGylated. TABLE 3 GH EC₅₀ (nM) GH EC₅₀ (nM) WHO WT0.4 ± 0.1 (n = 8) G120R >200,000 N-6His WT 0.6 ± 0.3 (n = 3)G120pAF >200,000 rat GH WT >200,000 G131pAF 0.8 ± 0.5 (n = 3) Y35pAF 0.7± 0.2 (n = 4) P133pAF 1.0 E88pAF 0.9 R134pAF 0.9 ± 0.3 (n = 4) Q91pAF2.0 ± 0.6 (n = 2) T135pAF 0.9 F92pAF 0.8 ± 0.4 (n = 9) G136pAF 1.4R94pAF 0.7 F139pAF 3.3 S95pAF 16.7 ± 1.0 (n = 2)  K140pAF 2.7 ± 0.9 (n =2) N99pAF 8.5 Y143pAF 0.8 ± 0.3 (n = 3) Y103pAF 130,000 K145pAF 0.6 ±0.2 (n = 3) Y111pAF 1.0 A155pAF 1.3

Example 3

This example details introduction of a carbonyl-containing amino acidand subsequent reaction with an aminooxy-containing PEG.

This Example demonstrates a method for the generation of a hGHpolypeptide that incorporates a ketone-containing non-naturally encodedamino acid that is subsequently reacted with an aminooxy-containing PEGof approximately 5,000 MW. Each of the residues 35, 88, 91, 92, 94, 95,99, 101, 103, 111, 120, 131, 133, 134, 135, 136, 139, 140, 143, 145, and155 identified according to the criteria of Example 1 (hGH) isseparately substituted with a non-naturally encoded amino acid havingthe following structure:

The sequences utilized for site-specific incorporation ofp-acetyl-phenylalanine into hGH are SEQ ID NO: 2 (hGH), and SEQ ID NO: 4(muttRNA, M. jannaschii mtRNA_(CUA) ^(Tyr)), and 16, 17 or 18 (TyrRSLW1, 5, or 6) described in Example 2 above.

Once modified, the hGH polypeptide variant comprising thecarbonyl-containing amino acid is reacted with an aminooxy-containingPEG derivative of the form:R—PEG(N)—O—(CH₂)_(n)—O—NH₂where R is methyl, n is 3 and N is approximately 5,000 MW. The purifiedhGH containing p-acetylphenylalanine dissolved at 10 mg/mL in 25 mM MES(Sigma Chemical, St. Louis, Mo.) pH 6.0, 25 mM Hepes (Sigma Chemical,St. Louis, Mo.) pH 7.0, or in 10 mM Sodium Acetate (Sigma Chemical, St.Louis, Mo.) pH 4.5, is reacted with a 10 to 100-fold excess ofaminooxy-containing PEG, and then stirred for 10-16 hours at roomtemperature (Jencks, W. J. Am. Chem. Soc. 1959, 81, pp 475). The PEG-hGHis then diluted into appropriate buffer for immediate purification andanalysis.

Example 4

Conjugation with a PEG consisting of a hydroxylamine group linked to thePEG via an amide linkage.

A PEG reagent having the following structure is coupled to aketone-containing non-naturally encoded amino acid using the proceduredescribed in Example 3:R—PEG(N)—O—(CH₂)₂—NH—C(O)(CH₂)_(n)—O—NH₂where R=methyl, n=4 and N is approximately 20,000 MW. The reaction,purification, and analysis conditions are as described in Example 3.

Example 5

This example details the introduction of two distinct non-naturallyencoded amino acids into hGH polypeptides.

This example demonstrates a method for the generation of a hGHpolypeptide that incorporates non-naturally encoded amino acidcomprising a ketone functionality at two positions among the followingresidues: E30, E74, Y103, K38, K41, K140, and K145. The hGH polypeptideis prepared as described in Examples 1 and 2, except that the selectorcodon is introduced at two distinct sites within the nucleic acid.

Example 6

This example details conjugation of hGH polypeptide to ahydrazide-containing PEG and subsequent in situ reduction.

A hGH polypeptide incorporating a carbonyl-containing amino acid isprepared according to the procedure described in Examples 2 and 3. Oncemodified, a hydrazide-containing PEG having the following structure isconjugated to the hGH polypeptide:R—PEG(N)—O—(CH₂)₂—NH—C(O)(CH₂)_(n)—X—NH—NH₂where R=methyl, n=2 and N=10,000 MW and X is a carbonyl (C═O) group. Thepurified hGH containing p-acetylphenylalanine is dissolved at between0.1-10 mg/mL in 25 mM MES (Sigma Chemical, St. Louis, Mo.) pH 6.0, 25 mMHepes (Sigma Chemical, St. Louis, Mo.) pH 7.0, or in 10 mM SodiumAcetate (Sigma Chemical, St. Louis, Mo.) pH 4.5, is reacted with a 1 to100-fold excess of hydrazide-containing PEG, and the correspondinghydrazone is reduced in situ by addition of stock 1M NaCNBH₃ (SigmaChemical, St. Louis, Mo.), dissolved in H₂O, to a final concentration of10-50 mM. Reactions are carried out in the dark at 4° C. to RT for 18-24hours. Reactions are stopped by addition of 1 M Tris (Sigma Chemical,St. Louis, Mo.) at about pH 7.6 to a final Tris concentration of 50 mMor diluted into appropriate buffer for immediate purification.

Example 7

This example details introduction of an alkyne-containing amino acidinto a hGH polypeptide and derivatization with mPEG-azide.

The following residues, 35, 88, 91, 92, 94, 95, 99, 101, 131, 133, 134,135, 136, 140, 143, 145, and 155, are each substituted with thefollowing non-naturally encoded amino acid (hGH; SEQ ID NO: 2):

The sequences utilized for site-specific incorporation ofp-propargyl-tyrosine into hGH are SEQ ID NO: 2 (hGH), SEQ ID NO: 4(muttRNA, M. jannaschii mtRNA_(CUA) ^(Tyr)), and 9, 10 or 11 describedin Example 2 above. The hGH polypeptide containing the propargyltyrosine is expressed in E. coli and purified using the conditionsdescribed in Example 3.

The purified hGH containing propargyl-tyrosine dissolved at between0.1-10 mg/mL in PB buffer (100 mM sodium phosphate, 0.15 M NaCl, pH=8)and a 10 to 1000-fold excess of an azide-containing PEG is added to thereaction mixture. A catalytic amount of CuSO₄ and Cu wire are then addedto the reaction mixture. After the mixture is incubated (including butnot limited to, about 4 hours at room temperature or 37° C., orovernight at 4° C.), H₂O is added and the mixture is filtered through adialysis membrane. The sample can be analyzed for the addition,including but not limited to, by similar procedures described in Example3.

In this Example, the PEG will have the following structure:R—PEG(N)—O—(CH₂)₂—NH—C(O)(CH₂)_(n)—N₃where R is methyl, n is 4 and N is 10,000 MW.

Example 8

This example details substitution of a large, hydrophobic amino acid ina hGH polypeptide with propargyl tyrosine.

A Phe, Trp or Tyr residue present within one the following regions ofhGH: 1-5 (N-terminus), 6-33 (A helix), 34-74 (region between A helix andB helix, the A-B loop), 75-96 (B helix), 97-105 (region between B helixand C helix, the B-C loop), 106-129 (C helix), 130-153 (region between Chelix and D helix, the C-D loop), 154-183 (D helix), 184-191(C-terminus) (SEQ ID NO: 2), is substituted with the followingnon-naturally encoded amino acid as described in Example 7:

Once modified, a PEG is attached to the hGH polypeptide variantcomprising the alkyne-containing amino acid. The PEG will have thefollowing structure:Me-PEG(N)—O—(CH₂)₂—N₃and coupling procedures would follow those in Example 7. This willgenerate a hGH polypeptide variant comprising a non-naturally encodedamino acid that is approximately isosteric with one of thenaturally-occurring, large hydrophobic amino acids and which is modifiedwith a PEG derivative at a distinct site within the polypeptide.

Example 9

This example details generation of a hGH polypeptide homodimer,heterodimer, homomultimer, or heteromultimer separated by one or morePEG linkers.

The alkyne-containing hGH polypeptide variant produced in Example 7 isreacted with a bifunctional PEG derivative of the form:N₃—(CH₂)_(n)—C(O)—NH—(CH₂)₂—O—PEG(N)—O—(CH₂)₂—NH—C(O)—(CH₂)_(n)—N₃where n is 4 and the PEG has an average MW of approximately 5,000, togenerate the corresponding hGH polypeptide homodimer where the two hGHmolecules are physically separated by PEG. In an analogous manner a hGHpolypeptide may be coupled to one or more other polypeptides to formheterodimers, homomultimers, or heteromultimers. Coupling, purification,and analyses will be performed as in Examples 7 and 3.

Example 10

This example details coupling of a saccharide moiety to a hGHpolypeptide.

One residue of the following is substituted with the non-naturallyencoded amino acid below: 29, 30, 33, 34, 35, 37, 39, 40, 49, 57, 59,66, 69, 70, 71, 74, 88, 91, 92, 94, 95, 98, 99, 101, 103, 107, 108, 111,122, 126, 129, 130, 131, 133, 134, 135, 136, 137, 139, 140, 141, 142,143, 145, 147, 154, 155, 156, 159, 183, 186, and 187 (hGH, SEQ ID NO: 2)as described in Example 3.

Once modified, the hGH polypeptide variant comprising thecarbonyl-containing amino acid is reacted with a β-linked aminooxyanalogue of N-acetylglucosamine (GlcNAc). The hGH polypeptide variant(10 mg/mL) and the aminooxy saccharide (21 mM) are mixed in aqueous 100mM sodium acetate buffer (pH 5.5) and incubated at 37° C. for 7 to 26hours. A second saccharide is coupled to the first enzymatically byincubating the saccharide-conjugated hGH polypeptide (5 mg/mL) withUDP-galactose (16 mM) and β-1,4-galacytosyltransferase (0.4 units/mL) in150 mM HEPES buffer (pH 7.4) for 48 hours at ambient temperature(Schanbacher et al. J. Biol. Chem. 1970, 245, 5057-5061).

Example 11

This example details generation of a PEGylated hGH polypeptideantagonist.

One of the following residues, 1, 2, 3, 4, 5, 8, 9, 11, 12, 15, 16, 19,22, 103, 109, 112, 113, 115, 116, 119, 120, 123, or 127 (hGH, SEQ ID NO:2 or the corresponding amino acids in SEQ ID NO: 1 or 3), is substitutedwith the following non-naturally encoded amino acid as described inExample 3.

Once modified, the hGH polypeptide variant comprising thecarbonyl-containing amino acid will be reacted with anaminooxy-containing PEG derivative of the form:R—PEG(N)—O—(CH₂)_(n)—O—NH₂where R is methyl, n is 4 and N is 20,000 MW to generate a hGHpolypeptide antagonist comprising a non-naturally encoded amino acidthat is modified with a PEG derivative at a single site within thepolypeptide. Coupling, purification, and analyses are performed as inExample 3.

Example 12 Generation of a hGH Polypeptide Homodimer, Heterodimer,Homomultimer, or Heteromultimer in which the hGH Molecules are LinkedDirectly

A hGH polypeptide variant comprising the alkyne-containing amino acidcan be directly coupled to another hGH polypeptide variant comprisingthe azido-containing amino acid, each of which comprise non-naturallyencoded amino acid substitutions at the sites described in, but notlimited to, Example 10. This will generate the corresponding hGHpolypeptide homodimer where the two hGH polypeptide variants arephysically joined at the site II binding interface. In an analogousmanner a hGH polypeptide polypeptide may be coupled to one or more otherpolypeptides to form heterodimers, homomultimers, or heteromultimers.Coupling, purification, and analyses are performed as in Examples 3, 6,and 7.

Example 13

The polyalkylene glycol (P—OH) is reacted with the alkyl halide (A) toform the ether (B). In these compounds, n is an integer from one to nineand R′ can be a straight- or branched-chain, saturated or unsaturatedC1, to C20 alkyl or heteroalkyl group. R′ can also be a C3 to C7saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, asubstituted or unsubstituted aryl or heteroaryl group, or a substitutedor unsubstituted alkaryl (the alkyl is a C₁ to C₂₀ saturated orunsaturated alkyl) or heteroalkaryl group. Typically, PEG-OH ispolyethylene glycol (PEG) or monomethoxy polyethylene glycol (mPEG)having a molecular weight of 800 to 40,000 Daltons (Da).

Example 14

mPEG-OH+Br—CH₂—C≡CH→mPEG-O—CH₂—C≡CH

mPEG-OH with a molecular weight of 20,000 Da (mPEG-OH 20 kDa; 2.0 g, 0.1mmol, Sunbio) was treated with NaH (12 mg, 0.5 mmol) in THF (35 mL). Asolution of propargyl bromide, dissolved as an 80% weight solution inxylene (0.56 mL, 5 mmol, 50 equiv., Aldrich), and a catalytic amount ofKI were then added to the solution and the resulting mixture was heatedto reflux for 2 hours. Water (1 mL) was then added and the solvent wasremoved under vacuum. To the residue was added CH₂Cl₂ (25 mL) and theorganic layer was separated, dried over anhydrous Na₂SO₄, and the volumewas reduced to approximately 2 mL. This CH₂Cl₂ solution was added todiethyl ether (150 mL) drop-wise. The resulting precipitate wascollected, washed with several portions of cold diethyl ether, and driedto afford propargyl-O-PEG.

Example 15

mPEG-OH+Br—(CH₂)₃—C≡CH→mPEG-O—(CH₂)₃—C≡CH

The mPEG-OH with a molecular weight of 20,000 Da (mPEG-OH 20 kDa; 2.0 g,0.1 mmol, Sunbio) was treated with NaH (12 mg, 0.5 mmol) in THF (35 mL).Fifty equivalents of 5-bromo-1-pentyne (0.53 mL, 5 mmol, Aldrich) and acatalytic amount of KI were then added to the mixture. The resultingmixture was heated to reflux for 16 hours. Water (1 mL) was then addedand the solvent was removed under vacuum. To the residue was addedCH₂Cl₂ (25 mL) and the organic layer was separated, dried over anhydrousNa₂SO₄, and the volume was reduced to approximately 2 mL. This CH₂Cl₂solution was added to diethyl ether (150 mL) drop-wise. The resultingprecipitate was collected, washed with several portions of cold diethylether, and dried to afford the corresponding alkyne. 5-chloro-1-pentynemay be used in a similar reaction.

Example 16

m-HOCH₂C₆H₄OH+NaOH+Br—CH₂—C≡CH→m-HOCH₂C₆H₄O—CH₂—C≡CH  (1)m-HOCH₂C₆H₄O—CH₂—C≡CH+MsCl+N(Et)₃ →m-MsOCH₂C₆H₄O—CH₂—C≡CH  (2)m-MsOCH₂C₆H₄O—CH₂—C≡CH+LiBr→m-Br—CH₂C₆H₄O—CH₂—C≡CH  (3)mPEG-OH+m-Br—CH₂C₆H₄O—CH₂—C≡CH→mPEG-O—CH₂—C₆H₄O—CH₂—C≡CH  (4)

To a solution of 3-hydroxybenzylalcohol (2.4 g, 20 mmol) in THF (50 mL)and water (2.5 mL) was first added powdered sodium hydroxide (1.5 g,37.5 mmol) and then a solution of propargyl bromide, dissolved as an 80%weight solution in xylene (3.36 mL, 30 mmol). The reaction mixture washeated at reflux for 6 hours. To the mixture was added 10% citric acid(2.5 mL) and the solvent was removed under vacuum. The residue wasextracted with ethyl acetate (3×15 mL) and the combined organic layerswere washed with saturated NaCl solution (10 mL), dried over MgSO₄ andconcentrated to give the 3-propargyloxybenzyl alcohol.

Methanesulfonyl chloride (2.5 g, 15.7 mmol) and triethylamine (2.8 mL,20 mmol) were added to a solution of compound 3 (2.0 g, 11.0 mmol) inCH₂Cl₂ at 0° C. and the reaction was placed in the refrigerator for 16hours. A usual work-up afforded the mesylate as a pale yellow oil. Thisoil (2.4 g, 9.2 mmol) was dissolved in THF (20 mL) and LiBr (2.0 g, 23.0mmol) was added. The reaction mixture was heated to reflux for 1 hourand was then cooled to room temperature. To the mixture was added water(2.5 mL) and the solvent was removed under vacuum. The residue wasextracted with ethyl acetate (3×15 mL) and the combined organic layerswere washed with saturated NaCl solution (10 mL), dried over anhydrousNa₂SO₄, and concentrated to give the desired bromide.

mPEG-OH 20 kDa (1.0 g, 0.05 mmol, Sunbio) was dissolved in THF (20 mL)and the solution was cooled in an ice bath. NaH (6 mg, 0.25 mmol) wasadded with vigorous stirring over a period of several minutes followedby addition of the bromide obtained from above (2.55 g, 11.4 mmol) and acatalytic amount of KI. The cooling bath was removed and the resultingmixture was heated to reflux for 12 hours. Water (1.0 mL) was added tothe mixture and the solvent was removed under vacuum. To the residue wasadded CH₂Cl₂ (25 mL) and the organic layer was separated, dried overanhydrous Na₂SO₄, and the volume was reduced to approximately 2 mL.Dropwise addition to an ether solution (150 mL) resulted in a whiteprecipitate, which was collected to yield the PEG derivative.

Example 17

mPEG-NH₂+X—C(O)—(CH₂)_(n)—C≡CR′→mPEG-NH—C(O)—(CH₂)_(n)—C≡CR′

The terminal alkyne-containing poly(ethylene glycol) polymers can alsobe obtained by coupling a poly(ethylene glycol) polymer containing aterminal functional group to a reactive molecule containing the alkynefunctionality as shown above. n is between 1 and 10. R′ can be H or asmall alkyl group from C1 to C4.

Example 18

HO₂C—(CH₂)₂—C═CH+NHS+DCC→NHSO—C(O)—(CH₂)₂—C≡CH  (1)mPEG-NH₂+NHSO—C(O)—(CH₂)₂—C≡CH→mPEG-NH—C(O)—(CH₂)₂—C≡CH  (2)

4-pentynoic acid (2.943 g, 3.0 mmol) was dissolved in CH₂Cl₂ (25 mL).N-hydroxysuccinimide (3.80 g, 3.3 mmol) and DCC (4.66 g, 3.0 mmol) wereadded and the solution was stirred overnight at room temperature. Theresulting crude NHS ester 7 was used in the following reaction withoutfurther purification.

mPEG-NH₂ with a molecular weight of 5,000 Da (mPEG-NH₂, 1 g, Sunbio) wasdissolved in THF (50 mL) and the mixture was cooled to 4° C. NHS ester 7(400 mg, 0.4 mmol) was added portion-wise with vigorous stirring. Themixture was allowed to stir for 3 hours while warming to roomtemperature. Water (2 mL) was then added and the solvent was removedunder vacuum. To the residue was added CH₂Cl₂ (50 mL) and the organiclayer was separated, dried over anhydrous Na₂SO₄, and the volume wasreduced to approximately 2 mL. This CH₂Cl₂ solution was added to ether(150 mL) drop-wise. The resulting precipitate was collected and dried invacuo.

Example 19

This Example represents the preparation of the methane sulfonyl ester ofpoly(ethylene glycol), which can also be referred to as themethanesulfonate or mesylate of poly(ethylene glycol). The correspondingtosylate and the halides can be prepared by similar procedures.mPEG-OH+CH₃SO₂Cl+N(Et)₃ →mPEG-O—SO₂CH₃ →mPEG-N₃

The mPEG-OH (MW=3,400, 25 g, 10 mmol) in 150 mL of toluene wasazeotropically distilled for 2 hours under nitrogen and the solution wascooled to room temperature. 40 mL of dry CH₂Cl₂ and 2.1 mL of drytriethylamine (15 mmol) were added to the solution. The solution wascooled in an ice bath and 1.2 mL of distilled methanesulfonyl chloride(15 mmol) was added dropwise. The solution was stirred at roomtemperature under nitrogen overnight, and the reaction was quenched byadding 2 mL of absolute ethanol. The mixture was evaporated under vacuumto remove solvents, primarily those other than toluene, filtered,concentrated again under vacuum, and then precipitated into 100 mL ofdiethyl ether. The filtrate was washed with several portions of colddiethyl ether and dried in vacuo to afford the mesylate.

The mesylate (20 g, 8 mmol) was dissolved in 75 ml of THF and thesolution was cooled to 4° C. To the cooled solution was added sodiumazide (1.56 g, 24 mmol). The reaction was heated to reflux undernitrogen for 2 hours. The solvents were then evaporated and the residuediluted with CH₂Cl₂ (50 mL). The organic fraction was washed with NaClsolution and dried over anhydrous MgSO₄. The volume was reduced to 20 mland the product was precipitated by addition to 150 ml of cold dryether.

Example 20

N₃—C₆H₄—CO₂H→N₃—C₆H₄—CH₂OH  (1)N₃—C₆H₄CH₂OH→Br—CH₂—C₆H₄—N₃  (2)mPEG-OH+Br—CH₂—C₆H₄—N₃ →mPEG-O—CH₂—C₆H₄—N₃  (3)

4-azidobenzyl alcohol can be produced using the method described in U.S.Pat. No. 5,998,595, which is incorporated by reference herein.Methanesulfonyl chloride (2.5 g, 15.7 mmol) and triethylamine (2.8 mL,20 mmol) were added to a solution of 4-azidobenzyl alcohol (1.75 g, 11.0mmol) in CH₂Cl₂ at 0° C. and the reaction was placed in the refrigeratorfor 16 hours. A usual work-up afforded the mesylate as a pale yellowoil. This oil (9.2 mmol) was dissolved in THF (20 mL) and LiBr (2.0 g,23.0 mmol) was added. The reaction mixture was heated to reflux for 1hour and was then cooled to room temperature. To the mixture was addedwater (2.5 mL) and the solvent was removed under vacuum. The residue wasextracted with ethyl acetate (3×15 mL) and the combined organic layerswere washed with saturated NaCl solution (10 mL), dried over anhydrousNa₂SO₄, and concentrated to give the desired bromide.

mPEG-OH 20 kDa (2.0 g, 0.1 mmol, Sunbio) was treated with NaH (12 mg,0.5 mmol) in THF (35 mL) and the bromide (3.32 g, 15 mmol) was added tothe mixture along with a catalytic amount of KI. The resulting mixturewas heated to reflux for 12 hours. Water (1.0 mL) was added to themixture and the solvent was removed under vacuum. To the residue wasadded CH₂Cl₂ (25 mL) and the organic layer was separated, dried overanhydrous Na₂SO₄, and the volume was reduced to approximately 2 mL.Dropwise addition to an ether solution (150 mL) resulted in aprecipitate, which was collected to yield mPEG-O—CH₂—C₆H₄—N₃.

Example 21

NH₂—PEG-O—CH₂CH₂CO₂H+N₃—CH₂CH₂CO₂—NHS→N₃—CH₂CH₂—C(O)NH—PEG-O—CH₂CH₂CO₂H

NH₂—PEG-O—CH₂CH₂CO₂H (MW 3,400 Da, 2.0 g) was dissolved in a saturatedaqueous solution of NaHCO₃ (10 mL) and the solution was cooled to 0° C.3-azido-1-N-hydroxysuccinimido propionate (5 equiv.) was added withvigorous stirring. After 3 hours, 20 mL of H₂O was added and the mixturewas stirred for an additional 45 minutes at room temperature. The pH wasadjusted to 3 with 0.5 NH₂SO₄ and NaCl was added to a concentration ofapproximately 15 wt %. The reaction mixture was extracted with CH₂Cl₂(100 mL×3), dried over Na₂SO₄ and concentrated. After precipitation withcold diethyl ether, the product was collected by filtration and driedunder vacuum to yield the omega-carboxy-azide PEG derivative.

Example 22

mPEG-OMs+HC≡CLi→mPEG-O—CH₂—CH₂—C≡C—H

To a solution of lithium acetylide (4 equiv.), prepared as known in theart and cooled to −78° C. in THF, is added dropwise a solution ofmPEG-OMs dissolved in THF with vigorous stirring. After 3 hours, thereaction is permitted to warm to room temperature and quenched with theaddition of 1 mL of butanol. 20 mL of H₂O is then added and the mixturewas stirred for an additional 45 minutes at room temperature. The pH wasadjusted to 3 with 0.5 N H₂SO₄ and NaCl was added to a concentration ofapproximately 15 wt %. The reaction mixture was extracted with CH₂Cl₂(100 mL×3), dried over Na₂SO₄ and concentrated. After precipitation withcold diethyl ether, the product was collected by filtration and driedunder vacuum to yield the 1-(but-3-ynyloxy)-methoxypolyethylene glycol(mPEG).

Example 23

The azide- and acetylene-containing amino acids were incorporatedsite-selectively into proteins using the methods described in L. Wang,et al., (2001), Science 292:498-500, J. W. Chin et al., Science301:964-7 (2003)), J. W. Chin et al., (2002), Journal of the AmericanChemical Society 124:9026-9027; J. W. Chin, & P. G. Schultz, (2002),Chem Bio Chem 3(11):1135-1137; J. W. Chin, et al., (2002), PNAS UnitedStates of America 99:11020-11024: and, L. Wang, & P. G. Schultz, (2002),Chem. Comm., 1:1-11. Once the amino acids were incorporated, thecycloaddition reaction was carried out with 0.01 mM protein in phosphatebuffer (PB), pH 8, in the presence of 2 mM PEG derivative, 1 mM CuSO₄,and ˜1 mg Cu-wire for 4 hours at 37° C.

Example 24

This example describes the synthesis of p-Acetyl-D,L-phenylalanine (pAF)and m-PEG-hydroxylamine derivatives.

The racemic pAF was synthesized using the previously described procedurein Zhang, Z., Smith, B. A. C., Wang, L., Brock, A., Cho, C. & Schultz,P. G., Biochemistry, (2003) 42, 6735-6746.

To synthesize the m-PEG-hydroxylamine derivative, the followingprocedures were completed. To a solution of (N-t-Boc-aminooxy)aceticacid (0.382 g, 2.0 mmol) and 1,3-Diisopropylcarbodiimide (0.16 mL, 1.0mmol) in dichloromethane (DCM, 70 mL), which was stirred at roomtemperature (RT) for 1 hour, methoxy-polyethylene glycol amine(m-PEG-NH₂, 7.5 g, 0.25 mmol, Mt. 30 K, from BioVectra) andDiisopropylethylamine (0.1 mL, 0.5 mmol) were added. The reaction wasstirred at RT for 48 hours, and then was concentrated to about 100 mL.The mixture was added dropwise to cold ether (800 mL). Thet-Boc-protected product precipitated out and was collected by filtering,washed by ether 3×100 mL. It was further purified by re-dissolving inDCM (100 mL) and precipitating in ether (800 mL) twice. The product wasdried in vacuum yielding 7.2 g (96%), confirmed by NMR and Nihydrintest.

The deBoc of the protected product (7.0 g) obtained above was carriedout in 50% TFA/DCM (40 mL) at 0° C. for 1 hour and then at RT for 1.5hour. After removing most of TFA in vacuum, the TFA salt of thehydroxylamine derivative was converted to the HCl salt by adding 4N HClin dioxane (1 mL) to the residue. The precipitate was dissolved in DCM(50 mL) and re-precipitated in ether (800 mL). The final product (6.8 g,97%) was collected by filtering, washed with ether 3×100 mL, dried invacuum, stored under nitrogen. Other PEG (5K, 20K) hydroxylaminederivatives were synthesized using the same procedure.

Example 25

This example describes expression and purification methods used for hGHpolypeptides comprising a non-natural amino acid. Host cells have beentransformed with orthogonal tRNA, orthogonal aminoacyl tRNA synthetase,and hGH constructs.

A small stab from a frozen glycerol stock of the transformed DH10B(fis3)cells were first grown in 2 ml defined medium (glucose minimal mediumsupplemented with leucine, isoleucine, trace metals, and vitamins) with100 μg/ml ampicillin at 37° C. When the OD₆₀₀ reached 2-5, 60 μl wastransferred to 60 ml fresh defined medium with 100 μg/ml ampicillin andagain grown at 37° C. to an OD₆₀₀ of 2-5. 50 ml of the culture wastransferred to 2 liters of defined medium with 100 μg/ml ampicillin in a5 liter fermenter (Sartorius BBI). The fermenter pH was controlled at pH6.9 with potassium carbonate, the temperature at 37° C., the air flowrate at 5 lpm, and foam with the polyalkylene defoamer KFO F119(Lubrizol). Stirrer speeds were automatically adjusted to maintaindissolved oxygen levels ≧30% and pure oxygen was used to supplement theair sparging if stirrer speeds reached their maximum value. After 8hours at 37° C., the culture was fed a 50× concentrate of the definedmedium at an exponentially increasing rate to maintain a specific growthrate of 0.15 hour⁻¹. When the OD₆₀₀ reached approximately 100, a racemicmixture of para-acetyl-phenylalanine was added to a final concentrationof 3.3 mM, and the temperature was lowered to 28° C. After 0.75 hour,isopropyl-b-D-thiogalactopyranoside was added to a final concentrationof 0.25 mM. Cells were grown an additional 8 hour at 28° C., pelleted,and frozen at −80° C. until further processing.

The His-tagged mutant hGH proteins were purified using the ProBondNickel-Chelating Resin (Invitrogen, Carlsbad, Calif.) via the standardHis-tagged protein purification procedures provided by Invitrogen'sinstruction manual, followed by an anion exchange column.

The purified hGH was concentrated to 8 mg/ml and buffer exchanged to thereaction buffer (20 mM sodium acetate, 150 mM NaCl, 1 mM EDTA, pH 4.0).MPEG-Oxyamine powder was added to the hGH solution at a 20:1 molar ratioof PEG:hGH. The reaction was carried out at 28° C. for 2 days withgentle shaking. The PEG-hGH was purified from un-reacted PEG and hGH viaan anion exchange column.

The quality of each PEGylated mutant hGH was evaluated by three assaysbefore entering animal experiments. The purity of the PEG-hGH wasexamined by running a 4-12% acrylamide NuPAGE Bis-Tris gel with MES SDSrunning buffer under non-reducing conditions (Invitrogen). The gels werestained with Coomassie blue. The PEG-hGH band was greater than 95% purebased on densitometry scan. The endotoxin level in each PEG-hGH wastested by a kinetic LAL assay using the KTA² kit from Charles RiverLaboratories (Wilmington, Mass.), and it was less than 5 EU per dose.The biological activity of the PEG-hGH was assessed with the IM-9 pSTAT5bioassay (mentioned in Example 2), and the EC₅₀ value was less than 15nM.

Example 26

This example describes methods for evaluating purification andhomogeneity of hGH polypeptides comprising a non-natural amino acid.

FIG. 8 is a SDS-PAGE of hGH polypeptides comprising a non-natural aminoacid at position 92. Lanes 3, 4, and 5 of the gel show hGH comprising ap-acetyl-phenylalanine at position 92 covalently linked to either a 5kDa, 20 kDa, or 30 kDa PEG molecule. Additional hGH polypeptidescomprising a non-natural amino acid that is PEGylated are shown FIG. 11.Five μg of each PEG-hGH protein was loaded onto each SDS-PAGE. FIG. 11,Panel A: Lane 1, molecular weight marker; lane 2, WHO rhGH referencestandard (2 μg); lanes 3 and 7, 30 KPEG-F92pAF; lane 4, 30KPEG-Y35pAF;lane 5, 3OKPEG-R134pAF; lane 6, 2OKPEG-R134pAF; lane 8, WHO rhGHreference standard (20 μg). FIG. 11, Panel B: Lane 9, molecular weightmarker, lane 10, WHO rhGH reference standard (2 μg); lane 11, 30KPEG-F92pAF; lane 12, 30 KPEG-K145pAF; lane 13, 30 KPEG-Y143pAF; lane14, 30KPEG-G131pAF; lane 15, 30KPEG-F92pAF/G120R, lane 16 WHO rhGHreference standard (20 μg). FIG. 9 shows the biological activity ofPEGylated hGH polypeptides (5 kDa, 20 kDa, or 30 kDa PEG) in IM-9 cells;methods were performed as described in Example 2.

The purity of the hGH-PEG conjugate can be assessed by proteolyticdegradation (including but not limited to, trypsin cleavage) followed bymass spectrometry analysis. Pepinsky R B., et al., J. Pharmcol. & Exp.Ther. 297(3):1059-66 (2001). Methods for performing tryptic digests arealso described in the European Pharmacopoeia (2002) 4^(th) Edition, pp.1938). Modifications to the methods described were performed. Samplesare dialyzed overnight in 50 mM TRIS-HCl, pH 7.5. rhGH polypeptides wereincubated with trypsin (TPCK-treated trypsin, Worthington) at a massratio of 66:1 for 4 hours in a 37° C. water bath. The samples wereincubated on ice for several minutes to stop the digestion reaction andsubsequently maintained at 4° C. during HPLC analysis. Digested samples(˜200 μg) were loaded onto a 25×0.46 cm Vydac C-8 column (5 μm beadsize, 100 Å pore size) in 0.1% trifluoroacetic acid and eluted with agradient from 0 to 80% acetonitrile over 70 min at a flow rate of 1ml/min at 30° C. The elution of tryptic peptides was monitored byabsorbance at 214 nm.

FIG. 10, Panel A depicts the primary structure of hGH with the trypsincleavage sites indicated and the non-natural amino acid substitution,F92pAF, specified with an arrow (Figure modified from Becker et al.Biotechnol Appl Biochem. (1988) 10(4):326-337). Panel B showssuperimposed tryptic maps of peptides generated from a hGH polypeptidecomprising a non-naturally encoded amino acid that is PEGylated (30K PEGHis₆-F92pAF rhGH, labeled A), peptides generated from a hGH polypeptidecomprising a non-naturally encoded amino acid (His₆-F92pAF rhGH, labeledB), and peptides generated from wild type hGH (WHO rhGH, labeled C).Comparison of the tryptic maps of WHO rhGH and His₆-F92pAF rhGH revealsonly two peak shifts, peptide peak 1 and peptide peak 9, and theremaining peaks are identical. These differences are caused by theaddition of the His₆ on the N-terminus of the expressed His₆-F92pAFrhGH, resulting in peak 1 shifting; whereas the shift in peak 9 iscaused by the substitution of phenylalanine at residue 92 withp-acetyl-phenylalanine. Panel C—A magnification of peak 9 from Panel Bis shown. Comparison of the His₆-F92pAF and the 30K PEG His₆-F92pAF rhGHtryptic maps reveals the disappearance of peak 9 upon pegylation ofHis₆-F92pAF rhGH, thus confirming that modification is specific topeptide 9.

Example 27

This example describes a homodimer formed from two hGH polypeptides eachcomprising a non-natural amino acid.

FIG. 12 compares IM-9 assay results from a His-tagged hGH polypeptidecomprising a p-acetyl-phenylalanine substitution at position 92 with ahomodimer of this modified polypeptide joined with a linker that isbifunctional having functional groups and reactivity as described inExample 25 for PEGylation of hGH.

Example 28

This example describes a monomer and dimer hGH polypeptide that act as ahGH antagonist.

An hGH mutein in which a G120R substitution has been introduced intosite II is able to bind a single hGH receptor, but is unable to dimerizetwo receptors. The mutein acts as an hGH antagonist in vitro, presumablyby occupying receptor sites without activating intracellular signalingpathways (Fuh, G., et al., Science 256:1677-1680 (1992)). FIG. 13, PanelA shows IM-9 assay data measuring phosphorylation of pSTAT5 by hGH withthe G120R substitution. A hGH polypeptide with a non-natural amino acidincorporated at the same position (G120) resulted in a molecule thatalso acts as an hGH antagonist, as shown in FIG. 13, Panel B. A dimer ofthe hGH antagonist shown in FIG. 13, Panel B was constructed joined witha linker that is bifunctional having functional groups and reactivity asdescribed in Example 25 for PEGylation of hGH. FIG. 14 shows that thisdimer also lacks biological activity in the IM-9 assay.

Additional assays were performed comparing hGH polypeptide comprising aG120pAF substitution with a dimer of G120pAF modified hGH polypeptidesjoined by a PEG linker. WHO hGH induced phosphorylation of STAT5 wascompeted with a dose-response range of the monomer and the dimer joinedby a PEG linker. Surface receptor competition studies were alsoperformed showing that the monomer and the dimer compete with GH forcell surface receptor binding on IM-9 and rat GHR (L43R)/BAF3 cells. Thedimer acted as a more potent antagonist than the monomer. Table 4 showsthe data from these studies. TABLE 4 Cell line Rat GHR IM-9 IM-9(L43R)/BAF3 Inhibition of Surface receptor Surface receptor pSTAT5competition competition Assay IC₅₀ (nM) IC₅₀ (nM) IC₅₀ (nM) G120pAFmonomer 3.3 8.4 3.1 (G120pAF) dimer, 0.7 2.7 1.4 PEG linker

Example 29

This example details the measurement of hGH activity and affinity of hGHpolypeptides for the hGH receptor.

Cloning and purification of rat GH receptor The extracellular domain ofrat GH receptor (GHR ECD, amino acids S29-T238) was cloned into pET20bvector (Novagen) between Nde I and Hind III sites in frame withC-terminal 6H is tag. A mutation of L43 to R was introduced to furtherapproximate the human GH receptor binding site (Souza et al., Proc NatlAcad Sci USA. (1995) 92(4): 959-63). Recombinant protein was produced inBL21 (DE3) E. coli cells (Novagen) by induction with 0.4 mM IPTG at 30°C. for 4-5 hours. After lysing the cells, the pellet was washed fourtimes by resuspending in a dounce with 30 mL of 50 mM Tris, pH 7.6, 100mM NaCl, 1 mM EDTA, 1% Triton X-100, and twice with the same bufferwithout Triton X-100. At this point inclusion bodies consisted of morethan 95% GHR ECD and were solubilized in 0.1M Tris, pH 11.5, 2M urea.Refolding was accomplished by means of passing an aliquot of theinclusion body solution through a S100 (Sigma) gel filtration column,equilibrated with 50 mM Tris, pH 7.8, 1 M L-arginine, 3.7 mM cystamine,6.5 mM cysteamine. Fractions containing soluble protein were combinedand dialyzed against 50 mM Tris, pH 7.6, 200 mM NaCl, 10% glycerol. Thesample was briefly centrifuged to remove any precipitate and incubatedwith an aliquot of Talon resin (Clontech), according to manufacturer'sinstructions. After washing the resin with 20 volumes of dialysis buffersupplemented with 5 mM imidazole, protein was eluted with 120 mMimidazole in dialysis buffer. Finally, the sample was dialyzed overnightagainst 50 mM Tris, pH 7.6, 30 mM NaCl, 1 mM EDTA, 10% glycerol,centrifuged briefly to remove any precipitate, adjusted to 20% glycerolfinal concentration, aliquoted and stored at −80 C. Concentration of theprotein was measured by OD(280) using calculated extinction coefficientof ε=65,700 M⁻¹*cm⁻¹.

Biocore™ Analysis of Binding of GH to GHR

Approximately 600-800 RUs of soluble GHR ECD was immobilized on aBiacore™ CM5 chip, using a standard amine-coupling procedure, asrecommended by the manufacturer. Even though a significant portion ofthe receptor was inactivated by this technique, it was foundexperimentally that this level of immobilization was sufficient toproduce maximal specific GH binding response of about 100-150 RUs, withno noticeable change in binding kinetics. See, e.g., Cunningham et al. JMol. Biol. (1993) 234(3): 554-63 and Wells J A. Proc Natl Acad Sci USA(1996) 93(1): 1-6).

Various concentrations of wild type or mutant GH (0.1-300 nM) in HBS-EPbuffer (Biacore™, Pharmacia) were injected over the GHR surface at aflow rate of 401l/min for 4-5 minutes, and dissociation was monitoredfor 15 minutes post-injection. The surface was regenerated by a 15second pulse of 4.5M MgCl₂. Only a minimal loss of binding affinity(1-5%) was observed after at least 100 regeneration cycles. Referencecell with no receptor immobilized was used to subtract any buffer bulkeffects and non-specific binding.

Kinetic binding data obtained from GH titration experiments wasprocessed with BiaEvaluation 4.1 software (BIACORE™). “Bivalent analyte”association model provided satisfactory fit (chi² values generally below3), in agreement with proposed sequential 1:2 (GH:GHR) dimerization(Wells J A. Proc Natl Acad Sci USA (1996) 93(1): 1-6). Equilibriumdissociation constants (Kd) were calculated as ratios of individual rateconstants (k_(off)/k_(on)).

Table 5 indicates the binding parameters from Biacore™ using rat GHR ECD(L43R) immobilized on a CM5 chip. TABLE 5 GH k_(on), ×10,⁻⁵ 1/M*sk_(off), ×10⁴, 1/s K_(d), nM WHO WT 6.4 3.8 0.6 N-6His WT 9 5.6 0.6 ratGH WT 0.33 83 250 N12pAF 12.5 4.6 0.4 R16pAF 6.8 4.8 0.7 Y35pAF 7.8 5.30.7 E88pAF 6.8 5.4 0.8 Q91pAF 6.6 4.9 0.7 F92pAF 8.6 5.0 0.6 R94pAF 5.66.0 1.1 S95pAF 0.7 3.1 4.3 N99pAF 2.2 3.8 1.7 Y103pAF ˜0.06 ˜6 >100Y111pAF 8.4 4.8 0.6 G120R 2.2 22 10 G120pAF 1.1 23 20 G131pAF 6.0 5.30.9 P133pAF 6.4 4.9 0.8 R134pAF 8.4 5.8 0.7 T135pAF 7.2 4.5 0.6 G136pAF6.2 4.3 0.7 F139pAF 6.8 4.4 0.7 K140pAF 7.2 3.7 0.5 Y143pAF 7.8 6.7 0.9K145pAF 6.4 5.0 0.8 A155pAF 5.8 4.4 0.8 F92pAF-5KD PEG 6.2 2.3 0.4F92pAF-20KD PEG 1.7 1.8 1.1 F92pAF-30KD PEG 1.3 0.9 0.7 R134pAF-5KD PEG6.8 2.7 0.4 R134pAF-30KD PEG 0.7 1.7 2.4 Y35pAF-30KD PEG 0.9 0.7 0.7(G120pAF) dimer 0.4 1.5 3.4 (F92pAF) dimer 3.6 1.8 0.5

The IL-3 dependent mouse cell line, BAF3, was routinely passaged in RPMI1640, sodium pyruvate, penicillin, streptomycin, 10% heat-inactivatedfetal calf serum, 50 uM 2-mercaptoethanol and 10% WEHI-3 cell lineconditioned medium as source of IL-3. All cell cultures were maintainedat 37° C. in a humidified atmosphere of 5% CO₂.

The BAF3 cell line was used to establish the rat GHR (L43R) stable cellclone, 2E2-2B12-F4. Briefly, 1×10⁷ mid-confluent BAF3 cells wereelectroporated with 15 ug of linearized pcDNA3.1 plasmid containing thefull length rat GHR (L43R) cDNA. Transfected cells were allowed torecover for 48 hours before cloning by limiting dilution in mediacontaining 800 ug/ml G418 and 5 mM WHO hGH. GHR expressing transfectantswere identified by surface staining with antibody against human GHR (R&DSystems, Minneapolis, Minn.) and analyzed on a FACS Array (BDBiosciences, San Diego, Calif.). Transfectants expressing a good levelof GHR were then screened for proliferative activity against WHO hGH ina BrdU proliferation assay (as described below). Stably transfected ratGHR (L43R) cell clones were established upon two further rounds ofrepeated subcloning of desired transfectants in the presence of 1.2mg/ml G418 and 5 nM hGH with constant profiling for surface receptorexpression and proliferative capability. Cell clone, 2E2-2B12-F4, thusestablished is routinely maintained in BAF3 media plus 1.2 mg/ml G418 inthe absence of hGH.

Proliferation by BrdU Labeling

Serum starved rat GHR (L43R) expressing BAF3 cell line, 2E2-2B12-F4,were plated at a density of 5×10⁴ cells/well in a 96-well plate. Cellswere activated with a 12-point dose range of hGH proteins and labeled atthe same time with 50 uM BrdU (Sigma, St. Louis, Mo.). After 48 hours inculture, cells were fixed/permeabilized with 100 ul of BDcytofix/cytoperm solution (BD Biosciences) for 30 min at roomtemperature. To expose BrdU epitopes, fixed/permeabilized cells weretreated with 30 ug/well of DNase (Sigma) for 1 hour at 37° C.Immunofluorescent staining with APC-conjugated anti-BrdU antibody (BDBiosciences) enabled sample analysis on the FACS Array.

Table 6 shows the bioactivity of PEG hGH mutants as profiled on thepSTAT5 (IM-9) and BrdU proliferation assays. WHO hGH is expressed asunity for comparison between assays. TABLE 6 pSTAT5 EC₅₀ ProliferationEC₅₀ hGH (nM) (nM) WHO WT 1.0 1.0 Y35pAF 1.3 1.6 ± 0.8 (n = 3)Y35pAF-30KPEG 10 5.4 ± 2.8 (n = 4) Y35pAF-40KPEG 53.3 24.0 + 11.0 (n =3) F92pAF 2.2 ± 0.4 (n = 9) 1.4 ± 0.7 (n = 4) F92pAF-5KPEG 5.1 + 0.4 (n= 3) ND F92pAF-20KPEG 10.5 + 0.8 (n = 3)  ND F92pAF-30KPEG 8.8 ± 1.2 (n= 8) 4.1 ± 0.9 (n = 3) F92pAF/G120R >200,000 >200,000F92pAF/G120R- >200,000 >200,000 30KPEG G131pAF 2.3 ± 1.8 (n = 2) 2.1 ±1.1 (n = 3) G131pAF-30KPEG 23.8 ± 1.7 (n = 2)  4.6 ± 2.4 (n = 3) R134pAF1.1 ± 0.2 (n = 2) 1.7 ± 0.3 (n = 3) R134pAF-20KPEG 5.3 ND R134pAF-30KPEG11.3 ± 1.1 (n = 2)  2.5 ± 0.7 (n = 4) Y143pAF 1.6 ± 0.1 (n = 2) 1.8 ±0.6 (n = 2) Y143pAF-30KPEG 12.3 ± 0.9 (n = 2)  6.6 ± 2.7 (n = 3) K145pAF2.3 ± 0.5 (n = 2) 3.0 ± 1.4 (n = 2) K145pAF-30KPEG 20.6 ± 9.8 (n = 2) 5.3 ± 3.5 (n = 3)

Example 30

This example describes methods to measure in vitro and in vivo activityof PEGylated hGH.

Cell Binding Assays

Cells (3×10⁶) are incubated in duplicate in PBS/1% BSA (100 μl) in theabsence or presence of various concentrations (volume: 10 μl) ofunlabeled GH, hGH or GM-CSF and in the presence of ¹²⁵I-GH (approx.100,000 cpm or 1 ng) at 0° C. for 90 minutes (total volume: 120 μl).Cells are then resuspended and layered over 200 μl ice cold FCS in a 350μl plastic centrifuge tube and centrifuged (1000 g; 1 minute). Thepellet is collected by cutting off the end of the tube and pellet andsupernatant counted separately in a gamma counter (Packard).

Specific binding (cpm) is determined as total binding in the absence ofa competitor (mean of duplicates) minus binding (cpm) in the presence of100-fold excess of unlabeled GH (non-specific binding). The non-specificbinding is measured for each of the cell types used. Experiments are runon separate days using the same preparation of ¹²⁵I-GH and shoulddisplay internal consistency. ¹²⁵I-GH demonstrates binding to the GHreceptor-producing cells. The binding is inhibited in a dose dependentmanner by unlabeled natural GH or hGH, but not by GM-CSF or othernegative control. The ability of hGH to compete for the binding ofnatural ¹²⁵I-GH, similar to natural GH, suggests that the receptorsrecognize both forms equally well.

In Vivo Studies of PEGylated hGH

PEG-hGH, unmodified hGH and buffer solution are administered to mice orrats. The results will show superior activity and prolonged half life ofthe PEGylated hGH of the present invention compared to unmodified hGHwhich is indicated by significantly increased bodyweight.

Measurement of the In Vivo Half-Life of Conjugated and Non-ConjugatedhGH and Variants Thereof.

All animal experimentation was conducted in an AAALAC accreditedfacility and under protocols approved by the Institutional Animal Careand Use Committee of St. Louis University. Rats were housed individuallyin cages in rooms with a 12-hour light/dark cycle. Animals were providedaccess to certified Purina rodent chow 5001 and water ad libitum. Forhypophysectomized rats, the drinking water additionally contained 5%glucose.

Pharmacokinetic Studies

The quality of each PEGylated mutant hGH was evaluated by three assaysbefore entering animal experiments. The purity of the PEG-hGH wasexamined by running a 4-12% acrylamide NuPAGE Bis-Tris gel with MES SDSrunning buffer under non-reducing conditions (Invitrogen, Carlsbad,Calif.). The gels were stained with Coomassie blue. The PEG-hGH band wasgreater than 95% pure based on densitometry scan. The endotoxin level ineach PEG-hGH was tested by a kinetic LAL assay using the KTA² kit fromCharles River Laboratories (Wilmington, Mass.), and was less than 5 EUper dose. The biological activity of the PEG-hGH was assessed with theIM-9 pSTAT5 bioassay (described in Example 2), and the EC₅₀ valueconfirmed to be less than 15 nM.

Pharmacokinetic properties of PEG-modified growth hormone compounds werecompared to each other and to nonPEGylated growth hormone in maleSprague-Dawley rats (261-425 g) obtained from Charles RiverLaboratories. Catheters were surgically installed into the carotidartery for blood collection. Following successful catheter installation,animals were assigned to treatment groups (three to six per group) priorto dosing. Animals were dosed subcutaneously with 1 mg/kg of compound ina dose volume of 0.41-0.55 ml/kg. Blood samples were collected atvarious time points via the indwelling catheter and into EDTA-coatedmicrofuge tubes. Plasma was collected after centrifugation, and storedat −80° C. until analysis. Compound concentrations were measured usingantibody sandwich growth hormone ELISA kits from either BioSourceInternational (Camarillo, Calif.) or Diagnostic Systems Laboratories(Webster, Tex.). Concentrations were calculated using standardscorresponding to the analog that was dosed. Pharmacokinetic parameterswere estimated using the modeling program WinNonlin (Pharsight, version4.1). Noncompartmental analysis with linear-up/log-down trapezoidalintegration was used, and concentration data was uniformly weighted.

FIG. 15 shows the mean (+/−S.D.) plasma concentrations following asingle subcutaneous dose in rats. Rats (n=3-4 per group) were given asingle bolus dose of 1 mg/kg hGH wild-type protein (WHO hGH), His-taggedhGH polypeptide (his-hGH), or His-tagged hGH polypeptide comprisingnon-natural amino acid p-acetyl-phenylalanine at position 92 covalentlylinked to 30 kDa PEG (30 KPEG-pAF92(his)hGH). Plasma samples were takenover the indicated time intervals and assayed for injected compound asdescribed. 30 KPEG-pAF92 (his)hGH has dramatically extended circulationcompared to control hGH.

FIG. 16 shows the mean (+/−S.D.) plasma concentrations following asingle subcutaneous dose in rats. Rats (n=3-6 per group) were given asingle bolus dose of 1 mg/kg protein. hGH polypeptides comprisingnon-natural amino acid p-acetyl-phenylalanine covalently linked to 30kDa PEG at each of six different positions were compared to WHO hGH and(his)-hGH. Plasma samples were taken over the indicated time intervalsand assayed for injected compound as described. Table 7 shows thepharmacokinetic parameter values for single-dose administration of hGHpolypeptides shown in FIG. 16. Concentration vs time curves wereevaluated by noncompartmental analysis (Pharsight, version 4.1). Valuesshown are averages (+/−standard deviation). C_(max): maximumconcentration; terminal t_(1/2): terminal half-life; AUC_(0->inf): areaunder the concentration-time curve extrapolated to infinity; MRT: meanresidence time; Cl/f: apparent total, plasma clearance; Vz/f: apparentvolume of distribution during terminal phase. TABLE 7 Pharmacokineticparameter values for single-dose 1 mg/kg bolus s.c. administration innormal male Sprague-Dawley rats. Parameter Cmax Terminal t_(1/2)AUC_(0−>inf) Cl/f Vz/f Compound (n) (ng/ml) (h) (ng × hr/ml) MRT (h)(ml/hr/kg) (ml/kg) WHO hGH (3) 529 (±127) 0.53 (±0.07) 759 (±178) 1.29(±0.05) 1,368 (±327) 1051 (±279) (his)hGH (4) 680 (±167) 0.61 (±0.05)1,033 (±92) 1.30 (±0.17) 974 (±84) 853 (±91) 30KPEG-pAF35(his)hGH (4)1,885 (±1,011) 4.85 (±0.80) 39,918 (±22,683) 19.16 (±4.00) 35 (±27) 268(±236) 30KPEG-pAF92(his)hGH (6) 663 (±277) 4.51 (±0.90) 10,539 (±6,639)15.05 (±2.07) 135 (±90) 959 (±833) 30KPEG-pAF131(his)hGH (5) 497 (±187)4.41 (±0.27) 6,978 (±2,573) 14.28 (±0.92) 161 (±61) 1,039 (±449)30KPEG-pAF134(his)hGH (3) 566 (±204) 4.36 (±0.33) 7,304 (±2,494) 12.15(±1.03) 151 (±63) 931 (±310) 30KPEG-pAF143(his)hGH (5) 803 (±149) 6.02(±1.43) 17,494 (±3,654) 18.83 (±1.59) 59 (±11) 526 (±213)30KPEG-pAF145(his)hGH (5) 634 (±256) 5.87 (±0.09) 13,162 (±6,726) 17.82(±0.56) 88 (±29) 743 (±252)Pharmacodynamic Studies

Hypophysectomized male Sprague-Dawley rats were obtained from CharlesRiver Laboratories. Pituitaries were surgically removed at 3-4 weeks ofage. Animals were allowed to acclimate for a period of three weeks,during which time bodyweight was monitored. Animals with a bodyweightgain of 0-8 g over a period of seven days before the start of the studywere included and randomized to treatment groups. Rats were administeredeither a bolus dose or daily dose subcutaneously. Throughout the studyrats were daily and sequentially weighed, anesthetized, bled, and dosed(when applicable). Blood was collected from the orbital sinus using aheparinized capillary tube and placed into an EDTA coated microfugetube. Plasma was isolated by centrifugation and stored at −80° C. untilanalysis.

FIG. 17 shows the mean (+/−S.D.) plasma concentrations following asingle subcutaneous dose in hypophysectomized rats. Rats (n=5-7 pergroup) were given a single bolus dose of 2.1 mg/kg protein. Results fromhGH polypeptides comprising non-natural amino acidp-acetyl-phenylalanine covalently linked to 30 kDa PEG at each of twodifferent positions (position 35, 92) are shown. Plasma samples weretaken over the indicated time intervals and assayed for injectedcompound as described.

The peptide IGF-1 is a member of the family of somatomedins orinsulin-like growth factors. IGF-1 mediates many of the growth-promotingeffects of growth hormone. IGF-1 concentrations were measured using acompetitive binding enzyme immunoassay kit against the providedrat/mouse IGF-1 standards (Diagnosic Systems Laboratories). Significantdifference was determined by t-test using two-tailed distribution,unpaired, equal variance. FIG. 18, Panel A shows the evaluation ofcompounds in hypophysectomized rats. Rats (n=5-7 per group) were giveneither a single dose or daily dose subcutaneously. Animals weresequentially weighed, anesthetized, bled, and dosed (when applicable)daily. Bodyweight results are shown for placebo treatments, wild typehGH (hGH), His-tagged hGH ((his)hGH), and hGH polypeptides comprisingp-acetyl-phenylalanine covalently-linked to 30 kDa PEG at positions 35and 92. FIG. 18, Panel B—A diagram is shown of the effect on circulatingplasma IGF-1 levels after administration of a single dose of hGHpolypeptides comprising a non-naturally encoded amino acid that isPEGylated. Bars represent standard deviation. In FIG. 18, Panel A, thebodyweight gain at day 9 for 30 KPEG-pAF35(his)hGH compound isstatistically different (p<0.0005) from the KPEG-pAF92(his)hGH compound,in that greater weight gain was observed.

FIG. 18, Panel C shows the evaluation of compounds in hypophysectomizedrats. Rats (n=11 per group) were given either a single dose or dailydose subcutaneously. Animals were sequentially weighed, anesthetized,bled, and dosed (when applicable) daily. Bodyweight results are shownfor placebo treatments, wild type hGH (hGH), and hGH polypeptidescomprising p-acetyl-phenylalanine covalently-linked to 30 kDa PEG atpositions 92, 134, 145, 131, and 143. FIG. 18, Panel D—A diagram isshown of the effect on circulating plasma IGF-1 levels afteradministration of a single dose of hGH polypeptides comprising anon-naturally encoded amino acid that is PEGylated (position 92, 134,145, 131, 143) compared to placebo treatments and wild type hGH. FIG.18, Panel E shows the mean (+/−S.D.) plasma concentrations correspondingto hGH polypeptides comprising a non-naturally encoded amino acid thatis PEGylated (position 92, 134, 145, 131, 143). Plasma samples weretaken over the indicated time intervals and assayed for injectedcompound as described. Bars represent standard deviation.

Example 31

Human Clinical Trial of the Safety and/or Efficacy of PEGylated hGHComprising a Non-Naturally Encoded Amino Acid.

Objective To compare the safety and pharmacokinetics of subcutaneouslyadministered PEGylated recombinant human hGH comprising a non-naturallyencoded amino acid with one or more of the commercially available hGHproducts (including, but not limited to Humatrope™ (Eli Lilly & Co.),Nutropin™ (Genentech), Norditropin™ (Novo-Nordisk), Genotropin™ (Pfizer)and Saizen/Serostim™ (Serono)).

Patients Eighteen healthy volunteers ranging between 20-40 years of ageand weighing between 60-90 kg are enrolled in the study. The subjectswill have no clinically significant abnormal laboratory values forhematology or serum chemistry, and a negative urine toxicology screen,HIV screen, and hepatitis B surface antigen. They should not have anyevidence of the following: hypertension; a history of any primaryhematologic disease; history of significant hepatic, renal,cardiovascular, gastrointestinal, genitourinary, metabolic, neurologicdisease; a history of anemia or seizure disorder; a known sensitivity tobacterial or mammalian-derived products, PEG, or human serum albumin;habitual and heavy consumer to beverages containing caffeine;participation in any other clinical trial or had blood transfused ordonated within 30 days of study entry; had exposure to hGH within threemonths of study entry; had an illness within seven days of study entry;and have significant abnormalities on the pre-study physical examinationor the clinical laboratory evaluations within 14 days of study entry.All subjects are evaluable for safety and all blood collections forpharmacokinetic analysis are collected as scheduled. All studies areperformed with institutional ethics committee approval and patientconsent.

Study Design This will be a Phase I, single-center, open-label,randomized, two-period crossover study in healthy male volunteers.Eighteen subjects are randomly assigned to one of two treatment sequencegroups (nine subjects/group). GH is administered over two separatedosing periods as a bolus s.c. injection in the upper thigh usingequivalent doses of the PEGylated hGH comprising a non-naturally encodedamino acid and the commercially available product chosen. The dose andfrequency of administration of the commercially available product is asinstructed in the package label. Additional dosing, dosing frequency, orother parameter as desired, using the commercially available productsmay be added to the study by including additional groups of subjects.Each dosing period is separated by a 14-day washout period. Subjects areconfined to the study center at least 12 hours prior to and 72 hoursfollowing dosing for each of the two dosing periods, but not betweendosing periods. Additional groups of subjects may be added if there areto be additional dosing, frequency, or other parameter, to be tested forthe PEGylated hGH as well. Multiple formulations of GH that are approvedfor human use may be used in this study. Humatrope™ (Eli Lilly & Co.),Nutropin™ (Genentech), Norditropin™ (Novo-Nordisk), Genotropin™ (Pfizer)and Saizen/Serostim™ (Serono)) are commercially available GH productsapproved for human use. The experimental formulation of hGH is thePEGylated hGH comprising a non-naturally encoded amino acid.

Blood Sampling Serial blood is drawn by direct vein puncture before andafter administration of hGH. Venous blood samples (5 mL) fordetermination of serum GH concentrations are obtained at about 30, 20,and 10 minutes prior to dosing (3 baseline samples) and at approximatelythe following times after dosing: 30 minutes and at 1, 2, 5, 8, 12, 15,18, 24, 30, 36, 48, 60 and 72 hours. Each serum sample is divided intotwo aliquots. All serum samples are stored at −20° C. Serum samples areshipped on dry ice. Fasting clinical laboratory tests (hematology, serumchemistry, and urinalysis) are performed immediately prior to theinitial dose on day 1, the morning of day 4, immediately prior to dosingon day 16, and the morning of day 19.

Bioanalytical Methods An ELISA kit procedure (Diagnostic SystemsLaboratory [DSL], Webster Tex.), is used for the determination of serumGH concentrations.

Safety Determinations Vital signs are recorded immediately prior to eachdosing (Days 1 and 16), and at 6, 24, 48, and 72 hours after eachdosing. Safety determinations are based on the incidence and type ofadverse events and the changes in clinical laboratory tests frombaseline. In addition, changes from pre-study in vital signmeasurements, including blood pressure, and physical examination resultsare evaluated.

Data Analysis Post-dose serum concentration values are corrected forpre-dose baseline GH concentrations by subtracting from each of thepost-dose values the mean baseline GH concentration determined fromaveraging the GH levels from the three samples collected at 30, 20, and10 minutes before dosing. Pre-dose serum GH concentrations are notincluded in the calculation of the mean value if they are below thequantification level of the assay. Pharmacokinetic parameters aredetermined from serum concentration data corrected for baseline GHconcentrations. Pharmacokinetic parameters are calculated by modelindependent methods on a Digital Equipment Corporation VAX 8600 computersystem using the latest version of the BIOAVL software. The followingpharmacokinetics parameters are determined: peak serum concentration(C_(max)); time to peak serum concentration (t_(max)); area under theconcentration-time curve (AUC) from time zero to the last blood samplingtime (AUC₀₋₇₂) calculated with the use of the linear trapezoidal rule;and terminal elimination half-life (t_(1/2)), computed from theelimination rate constant. The elimination rate constant is estimated bylinear regression of consecutive data points in the terminal linearregion of the log-linear concentration-time plot. The mean, standarddeviation (SD), and coefficient of variation (CV) of the pharmacokineticparameters are calculated for each treatment. The ratio of the parametermeans (preserved formulation/non-preserved formulation) is calculated.

Safety Results The incidence of adverse events is equally distributedacross the treatment groups. There are no clinically significant changesfrom baseline or pre-study clinical laboratory tests or blood pressures,and no notable changes from pre-study in physical examination resultsand vital sign measurements. The safety profiles for the two treatmentgroups should appear similar.

Pharmacokinetic Results Mean serum GH concentration-time profiles(uncorrected for baseline GH levels) in all 18 subjects after receivinga single dose of one or more of commercially available hGH products(including, but not limited to Humatrope™ (Eli Lilly & Co.), Nutropin™(Genentech), Norditropin™ (Novo-Nordisk), Genotropin™ (Pfizer) andSaizen/Serostim™ (Serono)) are compared to the PEGylated hGH comprisinga non-naturally encoded amino acid at each time point measured. Allsubjects should have pre-dose baseline GH concentrations within thenormal physiologic range. Pharmacokinetic parameters are determined fromserum data corrected for pre-dose mean baseline GH concentrations andthe C_(max) and t_(max) are determined. The mean t_(max) for theclinical comparator(s) chosen (Humatrope™ (Eli Lilly & Co.), Nutropin™(Genentech), Norditropin™ (Novo-Nordisk), Genotropin™ (Pfizer),Saizen/Serostim™ (Serono)) is significantly shorter than the t_(max) forthe PEGylated hGH comprising the non-naturally encoded amino acid.Terminal half-life values are significantly shorter for the commerciallyavailable hGH products tested compared with the terminal half-life forthe PEGylated hGH comprising a non-naturally encoded amino acid.

Although the present study is conducted in healthy male subjects,similar absorption characteristics and safety profiles would beanticipated in other patient populations; such as male or femalepatients with cancer or chronic renal failure, pediatric renal failurepatients, patients in autologous predeposit programs, or patientsscheduled for elective surgery. In conclusion, subcutaneouslyadministered single doses of PEGylated hGH comprising non-naturallyencoded amino acid will be safe and well tolerated by healthy malesubjects. Based on a comparative incidence of adverse events, clinicallaboratory values, vital signs, and physical examination results, thesafety profiles of the commercially available forms of hGH and PEGylatedhGH comprising non-naturally encoded amino acid will be equivalent. ThePEGylated hGH comprising non-naturally encoded amino acid potentiallyprovides large clinical utility to patients and health care providers.

Example 32

In the following Examples, ahGH and PEG ahGH are, respectively, hGH anda PEGylated hGH of SEQ ID NO:2 with a para-acetylphenylalanine at the 35position and, in the PEGylated hGH, the PEG linked via an oxime bondformed with the para-acetylphenylalanine, where the PEG is a linear 30kDa PEG.

STAT5 Phosphorylation Assay

The interaction of hGH with its receptor leads to the tyrosinephosphorylation of a signal transducer and activator of transcriptionfamily member, STAT5, in the human IM-9 lymphocyte cell line. Theconcentration of ^(a)hGH and PEG-^(a)hGH (required for 50% maximal STAT5phosphorylation (EC₅₀) was determined by activating IM-9 cells withincreasing concentrations of ^(a)hGH and PEG-^(a)hGH followed byintra-cellular immunofluorescent staining for phospho-STAT5. With theEC₅₀ of World Health Organization (WHO) standard hGH as 1.0, therelative EC₅₀ of ^(a)hGH as compared to WHO hGH was determined to be 1.1while that of PEG-^(a)hGH was 10.9.

Example 33 Proliferation Assay

PEG-^(a)hGH activity was determined in vitro by the use of a cell linethat proliferates in response to growth hormone. An example of such acell line is the rat GHR[L43R]/BAF3 cell clone named 2E2-2B12-F4, a linegenerated by stably transfecting mouse BAF3 cells with the rat GHRbearing a Leu to Arg substitution at position 43. The conversion ofLeu-43 of the rat receptor to Arg-43 renders the rat GHR more‘human-like’ and hence enables efficient hGH binding. Bromodeoxyuridine(BrdU)-labeling of actively dividing rat GHR[L43R]/BAF3 cells wasemployed to provide a high resolution, non-radioactive method for thequantitation of growth hormone induced proliferation. Setting the EC₅₀for WHO hGH as unity, the relative EC₅₀'s of ^(a)hGH and PEG-^(a)hGH ascompared to WHO hGH were determined to be 1.06±0.29 (n=11) and 5.37±1.23(n=9), respectively.

Example 34 Efficacy Studies of PEGylated hGH

Preclinical efficacy studies were conducted in a rat model of growthhormone deficiency. In this model, the pituitary gland was surgicallyremoved at approximately 4 weeks of age. The body weights of thesehypophysectomized rats were monitored after surgery and animals showingless than 7.5 grams of bodyweight gain over a period of 7 days beforethe study were deemed successfully hypophysectomized and were randomizedamongst the treatment groups. Animals showing more than 2.5 g ofweight-loss were deemed not healthy and were excluded from the study.The study was initiated approximately 3 weeks after the surgery.

Animals were dosed weekly (i.e. dosed on days 0 and 7) with eitherplacebo or increasing PEG-^(a)hGH dose. An additional treatment groupreceived Genotropin or placebo daily. Bodyweights and blood werecollected predose and daily. All animals were sacrificed on day 14. FIG.28 shows the plasma IGF-1 levels throughout the study. A robust,dose-dependent increase in IGF-1 was observed.

A dose-dependent increase in IGF-1 C_(max) was observed after each doseinterval. Curve fitting of the C_(max) values from the first doseestimated the minimum (Eo) and maximum (Emax) IGF-1 Cmax to be 136.7 and1,136.2 ng/mL respectively, and determined the ED₅₀ to be 0.136 mg/kg(Table 8).

Plasma IGF-1 levels also showed a clear dose-dependent increase whenexpressed as AUC. For this parameter, the AUC is expressed as AUC abovethe efficacious IGF-1 plasma level. Previous work showed that asustained IGF-1 concentration of 34 ng/mL above baseline resulted inbodyweight gain in hypophysectomized rats. In the current study, thebaseline IGF-1 level determined from the placebo group throughout thestudy was 128 ng/mL. An increase of 34 ng/mL over 128 ng/mL estimates anefficacious IGF-1 level of 162 ng/mL. Thus the AUC above 162 ng/mL wasintegrated. From this analysis, a dose-dependent increase in AUC aboveefficacious level was observed after each dose interval. Curve fittingof the AUC values from the first dose estimated the Eo and Emax IGF-1AUC to be 0 and 5,029.8 ng×hr/mL, respectively, and determined the ED₅₀for this parameter to be 0.909 mg/kg (Table 8)

The duration of IGF-1 levels induced above the estimated efficaciouslevel after the first dose was determined. At the higher doses, theIGF-1 levels had not returned to baseline before the second dose. Inthese instances, the IGF-1 concentration was extrapolated to theefficacious level using the calculated terminal slopes. From thisevaluation, a dose-dependent increase in duration of IGF-1 concentrationabove the efficacious level was evident. Curve fitting estimated minimumand maximum durations to be 0 and 8.84 days, respectively, anddetermined the ED₅₀ for this parameter to be 0.173 mg/kg (Table 8).

Bone growth was used as an additional pharmacodynamic measure tocalculate the PEG-^(a)hGH ED₅₀. Tibias were collected from each animalat the end of the study and immersion fixed in 10% neutral-bufferedformalin followed by radiography. The results are shown in FIG. 29.There was a clear dose-dependent increase in tibial bone length for thePEG-^(a)hGH treated animals. Moreover, there was a dose sparing effectcompared to the daily Genotropin treated animals. The amount ofGenotropin administered over a 7-day interval is equivalent to 2.1 mg/kgof PEG-^(a)hGH given weekly (i.e., days 0 and 7). An induction of tibialbone length increase similar to Genotropin is shown for PEG-^(a)hGH at0.42 to 1.0 mg/kg. This represents a ≧50% dose-sparing effect forPEG-^(a)hGH when compared to Genotropin.

PEG-^(a)hGH also induced a dose-dependent increase in bodyweight (FIG.30). Moreover, as with induction of tibial bone length, a dose-sparingeffect was evident for PEG-^(a)hGH over Genotropin. Daily treatment withGenotropin at 0.3 mg/kg/day induced 27.74 (+/−7.92) % bodyweight changeon day 14 from day 0. PEG-^(a)hGH, administered at the equivalent amountof GH (i.e. 2.1 mg/kg on days 0 and 7), induced greater bodyweightincrease over Genotropin. This dose of PEG-^(a)hGH resulted in 33.66(+/−7.55) % bodyweight change on day 14 from day 0. The percentbodyweight change induced by PEG-^(a)hGH administered at the lower doseof 1.0 mg/kg on days 0 and 7 induced 27.02 (+/−3.97) % bodyweight changeon day 14. This result represents a nearly 50% dose-sparing effect forPEG-^(a)hGH. Administration of unPEGylated GH weekly at 2.1 mg/kg dosefailed to induce body weight gain.

Curve fitting of the percent bodyweight change on day 14 estimated theminimum and maximum percent bodyweight change, induced by PEG-^(a)hGH,to be 7.17 and 50.65, respectively. From this curve, the ED₅₀ for thisparameter was determined to be 1.097 mg/kg (Table 8). TABLE 8 Efficacystudy results with PEG-^(a)hGH Pharmacodynamic Parameter Range of EffectED₅₀ (Effect) Treatment Regimen (Eo-Emax) (mg/kg) IGF-1 IGF-1 Cmax Aftersingle s.c. dose   136.7-1,136.2 0.136 Induction mg/mL IGF-1 AUC AboveAfter single s.c. dose    0-5,029.8 0.909 Efficacious Level ng × hr/mLDuration of IGF-1 After single s.c. dose   0-8.84 0.173 AboveEfficacious Level days Tibial Bone Length On day 14, after 2 30.52-33.670.424 weekly s.c. doses mm Percent Bodyweight Change On day 14, after 2  7.17-50.65% 1.097 weekly s.c. doses

The same plasma samples collected for IGF-1 concentration determinationwere also assayed for PEG-^(a)hGH concentration. The ELISA assay used isspecific for hGH, shows a reduced though still robust response forPEG-^(a)hGH, and does not detect rat GH. The PEG-^(a)hGH plasmaconcentration-time profiles are shown in FIG. 31. A dose-dependentincrease in PEG-^(a)hGH C_(max) occurred. The duration of PEG-^(a)hGHpersistence in the circulation, as well as the overall PEG-^(a)hGH AUC,increased in a dose-dependent manner. Genotropin, administered daily,was not detectable in plasma likely due to rapid clearance. ThePEG-^(a)hGH exposure results support the dose-dependent efficacy shownabove.

The hypophysectomized rat studies described above have shown thefollowing:

-   -   1. PEG-^(a)hGH dose-dependently increased IGF-1 plasma        concentration. IGF-1 C_(max) as well as IGF-1 AUC and duration        above efficacious level were all dose-dependently increased.    -   2. Tibial bone length and bodyweight were dose-dependently        increased.    -   3. Dose sparing of ≧50% over Genotropin administered daily was        demonstrated.    -   4. PEG-^(a)hGH showed dose-dependent increase in Cmax, AUC and        persistence in the circulation that correlated with the above        pharmacodynamic effects.

Example 35 Pharmacokinetic Studies

Rat Pharmacokinetics

Plasma concentration versus time profiles for PEG-^(a)hGH administeredsubcutaneously at various doses in the rat disease model are shown inExample 34, above.

Primate Pharmacokinetics

The pharmacokinetic properties of the version of PEG-^(a)hGH thatdiffers only in that the molecule contained a methionine at theN-terminal position (PEG-^(a)(met)hGH), were evaluated in malecynomolgus monkeys (FIG. 32).

A single injection of PEG-^(a)(met)hGH was administered either at asubcutaneous or intravenous dose of 0.75 mg/kg or 0.15 mg/kg,respectively. After subcutaneous administration, a Cmax of 4,977(+/−1,286) ng/mL was reached approximately 16 hours after injection. Theapparent terminal half-life was 12.23 (+/−1.72) hours, compared to 7.05(+/−0.47) in rat with PRG-^(a)hGH. The half-life after intravenousadministration was 6.42 (+/−0.51) hours. The dose-corrected AUC valueswere 362,443 and 312,440 ng×hr×kg/mL/mg for the subcutaneous andintravenous doses, respectively. The bioavailability from thesubcutaneous administration was calculated to be 116%.

Example 36 Rational for Dose and Dosing Regimen Proposed in the ClinicalProtocol

The optimal regimen for a PEGylated GH in humans is determined from thefollowing studies in animals.

Rat efficacy study—Dose-sparing (i.e. comparable efficacy to Genotropinbut with less PEG-^(a)hGH dose) observed in preclinical efficacy studiesare used in dose estimation required for biological activity in humans.

Pharmacokinetic evaluation in rat and cynomolgus monkey—Allometricscaling is used to estimate pharmacokinetic parameters in human.

Single-dose acute safety studies in rat and cynomolgus monkey are usedto evaluate exposure and determine NOAEL (no observed adverse effectlevel).

Rat one-month repeat-dose GLP safety study—Safety is evaluated followingone month of exposure from doses that are 2×, 6×, and 20× above themaximum recommended human dose (MRHD). MRHD=0.36 mg/kg/week forpediatric GHD.

Cynomolgus monkey one-month repeat-dose GLP safety study—Safety isevaluated following one month of exposure from doses that are 2×, 10×,and 30× above the maximum recommended human dose (MRHD). MRHD=0.36mg/kg/week for pediatric GHD.

Rat six-month repeat-dose GLP safety study. Safety is evaluatedfollowing six months of exposure with dosing such that the exposure inrats is 1-2× and 10× the observed exposure at the MRHD in humans.

Cynomolgus monkey six-month repeat-dose GLP safety study. Safety isevaluated following six months of exposure with dosing such that theexposure in monkeys is 1-2× and 10× the observed exposure at the MRHD inhumans.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons of ordinary skill in theart and are to be included within the spirit and purview of thisapplication and scope of the appended claims. All publications, patents,patent applications, and/or other documents cited in this applicationare incorporated by reference in their entirety for all purposes to thesame extent as if each individual publication, patent, patentapplication, and/or other document were individually indicated to beincorporated by reference for all purposes. TABLE 9 SEQUENCES CITED. SEQID # Sequence Name 1 Full-length amino acid sequence of hGH 2 The matureamino acid sequence of hGH (isoform 1) 3 The 20-kDa hGH variant in whichresidues 32-46 of hGH are deleted 21 Nucleotide Sequence for full lengthhGH 22 Nucleotide Sequence for mature hGH

1-79. (canceled)
 80. A method of increasing plasma concentration ofinsulin-like growth factor-1 (IGF-1) by administering a growth hormone(GH) composition, wherein said GH is linked to at least onewater-soluble polymer by an oxime bond.
 81. The method of claim 80wherein the GH is human growth hormone (hGH).
 82. The method of claim 81wherein the hGH comprises a sequence that is about 80% identical to SEQID NO:
 2. 83. The method of claim 80, wherein the GH comprises anon-naturally encoded amino acid.
 84. The method of claim 83 wherein thenon-naturally encoded amino acid comprises a ketone.
 85. The method ofclaim 84 wherein the non-naturally encoded amino acid ispara-acetylphenylalanine.
 86. The method of claim 80 wherein the oximebond is formed between a non-naturally encoded amino acid and awater-soluble polymer.
 87. The method of claim 80 wherein thewater-soluble polymer comprises polyethylene glycol (PEG).